Virus Research 92 (2003) 207 /212 www.elsevier.

com/locate/virusres

Review

Resistance mechanisms to plant viruses: an overview

Rob Goldbach *, Etienne Bucher, Marcel Prins
Laboratory of Virology, Wageningen University, Binnenhaven 11, PD-6709 Wageningen, The Netherlands Received 3 September 2002; received in revised form 3 December 2002; accepted 4 December 2002

Abstract To obtain virus-resistant host plants, a range of operational strategies can be followed nowadays. While for decades plant breeders have been able to introduce natural resistance genes in susceptible genotypes without knowing precisely what these resistance traits were, currently a growing number of (mostly) dominant resistance genes have been cloned and analyzed. This has led not only to a better understanding of the plants natural defence systems, but also opened the way to use these genes beyond species borders. Besides using natural resistance traits, also several novel, engineered forms of virus resistance have been developed over the past 15 years. The first successes were obtained embarking from the principle of pathogen-derived resistance (PDR) by transforming host plants with viral genes or sequences with the purpose to block a specific step during virus multiplication in the plant. As an unforeseen spin-off of these investments, the phenomenon of post-translational gene silencing (PTGS) was discovered, which to date is by far the most successful way to engineer resistance. It is generally believed that PTGS reflects a natural defence system of the plant, and part of the hypothesized components required for PTGS have been identified. As counteracting strategy, and confirming PTGS to be a natural phenomenon, a considerable number of viruses have acquired gene functions by which they can suppress PTGS. In addition to PDR and PTGS, further strategies for engineered virus resistance have been explored, including the use of pokeweed antiviral protein (PAP), 2?,5?-oligoadenylate synthetase and plantibodies. This paper will give a brief overview of the major strategies that have become operational during the past 10 years. # 2003 Elsevier Science B.V. All rights reserved.
Keywords: Pathogen-derived resistance; Hypersensitive response; Post-transcriptional gene silencing

1. Introduction As in many years of the past, plant viruses are still forming a major problem in the cultivation of many vegetable and ornamental crops throughout the world. For long times, these pathogens have been controlled using conventional measures like crop rotation and other cultivation techniques, early detection, destruction of infected source plants, cross-protection, breeding for resistance, and chemical control of their vectors (Bos, 2000; Hull, 2002). Increased knowledge of both the molecular genetics of plant viruses and, more recently, also of their hosts natural defence systems have resulted in the development of a number of novel ways to control virus diseases in plants. This paper presents a brief overview of novel insights in the natural defence mechanisms of plants to viruses as
* Corresponding author. E-mail address: rob.goldbach@wur.nl (R. Goldbach).

well as some technologies to control viruses by exploiting this knowledge. Special reference will be made throughout this paper to recent reviews in which these issues have been covered in a much more detailed way, and which are recommended to the reader for further reading.

2. Natural resistance to plant viruses Besides passive defence based on the presence of existing barriers like the rigid cell wall, plants exhibit active defence mechanisms upon recognition of pathogens such as viruses. The most common mechanism associated with active defence is the so-called hypersensitive response (HR), during which cells surrounding the primary infection site of the virus die due to a rapidly induced programmed cell death, which results in a visible necrotic local lesion. The induction of this response is preceded by a specific recognition of the

0168-1702/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1702(02)00353-2

208

R. Goldbach et al. / Virus Research 92 (2003) 207 /212

virus, and in many cases this is based on matching (dominant) gene products of the plant (produced from dominant resistance genes, R genes) and the virus (avirulence genes). To date a few dozen single dominant R resistance genes recognizing different categories of plant pathogens have been cloned and sequenced. Strikingly, despite the wide range of pathogen taxa (varying from virus, bacterium, fungi to nematode and insect pathogens), all R genes identified so far in a range of different model and crop species, encode proteins which can be categorized into only five classes (for a recent review, see Dangl and Jones, 2001). A few examples of R gene products are summarized in Table 1. The largest class of R genes encodes a so-called nucleotide-binding site plus leucine-rich repeat (NBLRR) type of protein. So far all R genes that have been isolated conferring resistance to viruses belong to this class. In other types of protein, LRR domains have been shown to function in protein /protein binding, peptide / ligand binding or protein /carbohydrate interaction (Jones and Jones, 1996; Kajava, 1998). It is tempting to assume that the R gene products directly or indirectly interact with other (host or virus-encoded) factors, but this still needs to be demonstrated. The same holds for the NB domain, which suggests these R gene products to bind GTP or ATP, the significance of which being not yet understood. Within the large class of NB-LRR resistance genes, a sub-division can be made on basis of the N-terminal domain. This can either be a (putative) coiled-coil (CC) domain (CC-NB-LRR), a leucine zipper domain (LZ-NB-LRR) or a so-called TIR domain, with homology to the intracellular signaling domains of the Drosophila Toll and the mammalian interleukin (IL)-1 receptors (TIR-NB-LRR). Clearly, functional studies need to be done to understand how these R gene products act, and trigger an effective response to, e.g. invading viruses. Comparative analysis of the growing number of R genes indicate that these have evolved from ancient gene families by duplication, subsequent mutation and recombination, as they are all
Table 1 Selected set of dominant resistance (R) genes against viruses and other pathogens R gene Prf Mi Rx HRT Sw -5 12c Bs2 Mla 1 N RPS4 Cf9 Plant Tomato Tomato Potato Arabidopsis Tomato Tomato Pepper Barley Tobacco Arabidopsis Tomato Pathogen P. syringae Meloidogyne incognita PVX TCV TSWV Fusarium oxysporum Xanthomonas campestris Blumeria graminis TMV P. syringae Cladosporium fulvum Type LZ-NB-LRR LZ-NB-LRR LZ-NB-LRR LZ-NB-LRR LZ-NB-LRR NB-LRR NB-LRR CC-NB-LRR TIR-NB-LRR TIR-NB-LRR LRR-TM

inter-related irrespective of which type of plant pathogen they act. An additional feature of R genes appears to be that they are always a part of a gene cluster of homologues, the function of these related homologues having mostly remained unclear to date. The availability of cloned R genes not only will lead to further unraveling of the working mechanisms of the natural defence systems in plants but also achieve their use across species borders. For a number of R genes, e.g. the tomato Sw -5 gene conferring resistance to a number of tospoviruses, it has been shown that it is functional in other host plants (Spassova et al., 2001).

3. Engineered resistance to plant viruses 3.1. Pathogen-derived resistance As (plant) viruses have small genomes, containing only a limited number of genes of which nowadays most functions during the viral replication cycle have been well elucidated, these pathogens are suitable targets for engineered resistance concepts based on the principle of pathogen-derived resistance (PDR). This concept was first launched by Sanford and Johnson (1985) as proposed broad application of pathogen-derived genes in generating specific host resistance. A pre-requisite for the use of PDR is of course that none of the approaches should interfere with essential host functions. Since viruses require replication of their genome and, encapsidation of their genome, spread from the original infection site through the host and through the environment, they encode specific genes essential for these processes, and these have been proven excellent candidates for developing host resistance based on PDR. Most successful and widely applied has been the exploitation of plant viral coat protein (CP) genes, thus achieving CP-mediated resistance. For a number of RNA viruses, including TMV, PVX, AlMV, CMV and TRV, it has been shown that host plants transgenically expressing their CP results in high levels of operational resistance (reviewed in a.o. Wilson, 1993; Beachy, 1994; Baulcombe, 1996), which can only be overcome at extremely high inoculation pressure or inoculation with unencapsidated viral RNA. The latter finding supported the idea that CP-mediated resistance is based on blocking the disassembly of the infecting virus due to the presence of transgenically expressed CP. Other successful approaches for achieving PDR have been the transformation of host plants with cDNA copies of symptom-suppressing satellites and with mutagenized movement protein (MP) or replicase subunits (Anderson et al., 1992; Lapidot et al., 1993; Audy et al., 1994; Baulcombe, 1994; Lomonossoff, 1995; Palukaitis and Zaitlin, 1997). Although the various strategies have been shown to be successful, under

R. Goldbach et al. / Virus Research 92 (2003) 207 /212

209

greenhouse and field conditions, so far transgenic crop plants expressing (parts of) functional genes or genome parts of plant viruses have not been widely introduced due to legislation problems related to biosafety issues. Indeed under laboratory conditions, it has been shown that recombination may occur between transgenically expressed viral RNA transcripts and infecting viral genomes (Greene and Allison, 1994; Canto et al., 2001). A success story worthwhile mentioning has been the control of papaya ringspot virus on Hawaii by the use of transgenic papaya (Ferreira et al., 2002). 3.2. Post-transcriptional gene silencing as mean to achieve virus resistance One of the major drawbacks of true PDR, i.e. a desired high level of transgenic expression of viral genes, has been overcome by introducing transgenic virus resistance based on co-suppression or post-transcriptional gene silencing (PTGS). PTGS, also known as RNA silencing refers to related processes of post-transcriptional control of gene expression found in plants and fungi, and more recently in some animal species, and involving suppression of foreign genetic elements such as viruses and transposons through a specific RNA breakdown mechanism. At least in plants, PTGS serves as an adaptive, antiviral defence system. PTGS was first discovered in transgenic petunia plants in which introduced gene copies of the chalcon synthetase gene together with the endogenous gene copy were co-ordinately (co-)suppressed (Van der Krol et al., 1990; Napoli et al., 1990). Next it was proposed by Lindbo et al. (1993) and Dougherty et al. (1994) as mechanism to explain a number of unexpected outcomes of experiments where virus resistance based on PDR was originally aimed. Indeed, it was frequently found in control experiments using untranslatable versions of viral genes (initially included to serve as negative controls) that similar levels
Table 2 Known silencing suppressing plant viruses Virus ACMV (Voinnet et al., 1999) BSMV (Yelina et al., 2002) BWYV (Pfeffer et al., 2002) CMV (Brigneti et al., 1998) Genus Begomovirus Hordeivirus Polerovirus Cucumovirus Suppression Protein AC2 gb P0 2b ? P15 P25 HcPro NS3 P1 P19 NSs

Complete Complete Complete Partial/systemic CPMV (Voinnet et al., 1999) Comovirus Partial PCV (Dunoyer et al., 2002) Pecluvirus Complete PVX (Voinnet et al., 2000) Potexvirus Partial/systemic PVY (Brigneti et al., 1998) Potyvirus Complete RHBV (Bucher et al., 2003) Tenuivirus Complete RYMV (Voinnet et al., Sobemovirus Complete 1999) TBSV (Voinnet et al., 1999) Tombusvirus Complete TSWV (Bucher et al., 2003) Tospovirus Complete

of transgenic resistance could be obtained. Moreover, it was frequently observed that transgenic plant lines, in which only low levels of transgenic viral RNA accumulated, exhibited the highest levels of resistance, while, instead, plant lines with high accumulating levels of transgenic RNA were poor performers. Further studies, in different systems, revealed that indeed in the resistant plant lines the transgenic RNA was rapidly broken down in a sequence-specific manner. Hence, transgenes with no sequence homology to the hosts genome can trigger RNA silencing and may do so in a very efficient way. To date transgenic resistance against virtually all major plant viruses, indiscriminate of the nature of their genome (RNA or DNA), has been reported. Although originally found in transgenic plants producing foreign or aberrant RNA, evidence has accumulated that PTGS in plants in fact acts as a natural antiviral defence system by surveying for aberrant RNAs such as double-stranded viral replication intermediates. In addition, the mechanism of a natural resistance gene was found to be based on RNA silencing (Covey et al., 1994). A very strong argument pointing toward the natural role of RNA silencing in antiviral defence is the presence of plant viral gene products that, in turn, suppress this silencing. The PTGS defence system acts remarkably efficient and works very broad, i.e. it can be activated in the plant against virtually any virus species. A simplified model to explain how PTGS results in virus resistance and how it can be enhanced by transgene expression is presented in Fig. 1. The model also accommodates data generated by genetic screens of Arabidopsis by which some factors involved in RNA silencing have been identified (Mourrain et al., 2000; Dalmay et al., 2000, 2001). Transgenically expressed ssRNA is first copied into short dsRNA during a surveillance/amplication process in which a host-encoded RNA-dependent RNA polymerase (RdRP) is involved. Such RdRPs have indeed been identified in various plant species (Schiebel et al., 1998), while genetic screens in Arabidopsis confirmed a crucial involvement in PTGS (in case of single copy sense transgenes). Transgenes with inverted repeats may directly produce dsRNA, without the involvement of an RdRP, while this is also true in case of an RNA virus infection where dsRNA is formed as replication intermediates. The dsRNA produced in either way form a trigger to recruit further components required for sequence-specific breakdown of the transgenic RNA, i.e. a Dicer-like dsRNAse, while ssRNA targets are broken down by the so-called RISC-like nuclease complexes, which also serve as sequence-specific targeting complexes due to the incorporation of 21 /23 nt short interfering RNAs (siRNAs). Hence, as a product of PTGS typically small, 21 /23 nt long RNA fragments are formed. These small interfering (si)RNAs probably also play a role in the systemic signal which is spread

210

R. Goldbach et al. / Virus Research 92 (2003) 207 /212

Fig. 1. General model for gene silencing in plants induced by viruses and transgenes. Cartoons of known and putatively involved genes are presented in the box with their homologues in plants, fungi, nematodes and insects.

through the plant. More recently, PTGS has been reported in animals, a.o. in nematodes (C. elegans ) and flies (Drosophila ), where it has been shown to be involved in genome defence against transposable elements, but where a possible antiviral role still needs to be substantiated (Fire et al., 1998; Kennerdell and Carthew, 1998; Plasterk, 2002). As plant-infecting viruses have to deal with PTGSbased defence mechanisms, it is not at all a surprise that these viruses can encode a counteracting activity, i.e. a suppressor of gene silencing. Table 2 gives an overview of viruses that have been shown to produce such a suppressor protein. Suppressors of silencing are often identified in silencing reversal assays, using silenced GFP transgenic plants (Brigneti et al., 1998; Voinnet et al., 1999; Dunoyer et al., 2002). One of the best studied suppressors of RNA silencing is the helper componentprotease (HCPro) encoded by potyviruses (see also the contribution of V. Vance in this issue). Distinct viruses may specify suppressors which target distinct steps in the silencing reaction, and which may act stronger or weaker on specific parts of the pathway. As already mentioned above, the finding that plant viruses encode such suppressors is consistent with the idea that RNA silencing operates as an innate antiviral defence mechanism, rather than being a mechanism to control host gene expression (for recent reviews, see Voinnet, 2001; Baulcombe, 2002).

As PTGS results in well-fragmented RNA, it might be argued that transgenic resistance against viruses based on this mechanism fulfils more easily the current high demands with respect to biosafety. Indeed PTGS-based resistance does not involve the transgenic production of functional viral genes or proteins, nor does it lead to the presence of transgenic RNA, which might become engaged in RNA recombination events. 3.3. Other strategies 3.3.1. Plantibodies The use of full size antibodies or antibody fragments can be an attractive addition to the palette of virus resistance strategies. Already in 1993, it has been demonstrated that transgenic expression of a singlechain (scFv) antibody against the CP of Artichoke mottle crinkle virus results in reduced susceptibility to the virus (Tavladoraki et al., 1993). The mechanism by which the antibody conferred resistance remained unanswered, but since this first publication different research groups have tried to optimize this approach, often referred to as plantibody technology, as high levels of expressed antibodies form a pre-requisite for success. Since 1993, only limited progress appears to have been made (Fecker et al., 1996; Schillberg et al., 2000), and this might have been due to a few bottlenecks intrinsic to the approach. First of all, high expression

R. Goldbach et al. / Virus Research 92 (2003) 207 /212

211

levels of protein should be reached. Second, the in planta expressed antibody should be targeted to the right intracellular compartment where the virus replication step that needs to be inhibited takes place. Some protein-stabilizing protocols have become available since, such as the addition of a KDEL retention tetrapeptide (Schouten et al., 1996; Conrad and Fiedler, 1998) as well as easy protocols to select promising antibodies, including the selection of mAbs using hybridoma technology and phage display libraries (for a recent review, see Schillberg et al., 2001). Furthermore, this may be the main bottleneck, antibodies should be selected which are able to block a crucial step in the virus multiplication or transmission cycle. Despite the early single success with a monoclonal recognizing a viral CP (Tavladoraki et al., 1993), this seems not easy, and prior to transforming plants with antibodies they should ideally be tested in a well-designed interference assay. Obviously, mAbs recognizing the viral polymerase or any other catalytically acting viral proteins are more promising than mAbs binding structural proteins. In this context, the work of Boonrod et al. (Abstract ICV) is worthwhile mentioning. 3.3.2. Pokeweed antiviral proteins A further strategy for introducing virus resistance in plants is the use of specific and natural inhibitors of virus replication. One promising category of candidate factors is the pokeweed antiviral proteins (PAPs) isolated from several organs of Phytolacca americana (Pokeweed). PAPs are single-chain ribosome-inactivating proteins (RIBs) and are therefore, upon application in antiviral protocols, potentially also toxic to the host plant (for reviews, see Wang and Tumer, 2000; Tumer et al., 1999; Van Damme et al., 2001; Nielsen and Boston, 2001). Various less toxic types and non-toxic forms have been found or engineered which confer resistance to viruses upon transgenic expression without the detrimental effects of ribosome inactivation (Wang et al., 1998; Zoubenko et al., 2000). 3.3.3. 2?,5?-Oligoadenylate synthetase During the 1990s, it has been tested whether the reconstruction of the mammalian 2?,5?-oligoadenylate (2-5A) pathway into the plant kingdom would achieve multiple virus resistance. Rather broad virus resistance was indeed obtained by transforming host plants with the mammalian 2-5A synthetase (Truve et al., 1993, 1994), but despite the great potential of this methodology it has not been further exploited since.

defence mechanisms of plants towards viruses have been obtained. By exploiting our increased knowledge of the molecular genetics of plant viruses and */not the least */the host plant, various antiviral strategies have successfully been developed over the past 15 years or so. Further exploitation of at least part of these strategies, however, seems to have hampered by growing societal concern, especially in Europe, with respect to the production of transgenic food. Therefore, it seems likely that in the near future two approaches will be further applied, i.e. the use of well-defined natural R genes, the functioning of which is expected to be fully unraveled within the next 10 years and which can be introduced very rapidly by marker-assisted breeding techniques, and the further exploitation of another type of natural defence, PTGS.

References
Anderson, J.M., Palukaitis, P., Zaitlin, M., 1992. A defective replicase gene induces resistance to cucumber mosaic virus in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA 89, 8759 /8763. Audy, P., Palukaitis, P., Slack, S.A., Zaitlin, M., 1994. Replicase mediated resistance to potato virus Y in transgenic plants. Mol. Plant Microbe Interact. 7, 15 /22. Baulcombe, D., 1994. Replicase-mediated resistance: a novel type of virus resistance in transgenic plants. Trends Microbiol. 2, 60 /63. Baulcombe, D.C., 1996. Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8 (10), 1833 /1844. Baulcombe, D., 2002. RNA silencing. Curr. Biol. 12 (3), 82 /84. Beachy, R.N., 1994. Introduction: transgenic resistance to plant viruses. Semin. Virol. 4, 327 /328. Bos, L., 2000. Plant Viruses, Unique and Intriguing Pathogens. Backhuys Publishers, Leiden, p. 358. Brigneti, G., Voinnet, O., Li, W.X., Ji, L.H., Ding, S.W., Baulcombe, D.C., 1998. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana . EMBO J. 17 (22), 6739 /6746. Bucher, E., Sijen, T., De Haan, P., Goldbach, R. and Prins, M., 2003. Negative strand Tospoviruses and Tenuiviruses encode a suppressor of gene silencing on analogous genomic positions. J. Virol. 77 (2), 1329 /1336. Canto, T., Choi, S.K., Palukaitis, P., 2001. A subpopulation of cucumber mosaic virus RNA 1 contains 3? termini originating from RNAs 2 or 3. J. Gen. Virol. 82, 941 /945. Conrad, U., Fiedler, U., 1998. Compartment-specic accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Mol. Biol. 38, 101 /109. Covey, S.N., Al-Kaff, N.S., Langara, A., Turner, D.S. 1997. Plants combat infections by gene silencing. Nature 385 (6619), 781 /782. Dalmay, T., Hamilton, A., Rudd, S., Angell, S., Baulcombe, D.C., 2000. An RNA-dependent RNA polymerase gene in Arabidopsis is required for post-transcriptional gene silencing mediated by a transgene but not by a virus. Cell 101 (5), 543 /553. Dalmay, T., Horseeld, R., Braunstein, T.H., Baulcombe, D.C., 2001. SDE3 encodes an RNA helicase required for post-transcriptional gene silencing in Arabidopsis. EMBO J. 20 (8), 2069 /2077. Dangl, J.L., Jones, J.D.G., 2001. Plant pathogens and integrated defence responses to infection. Nature 411, 826 /833. Dougherty, W.G., Lindbo, J.A., Smith, H.A., Parks, T.D., Swaney, S., Proebsting, W.M., 1994. RNA-mediated virus resistance in trans-

4. Conclusions By detailed studies on virus /plant interactions using novel technologies, increased insight in the natural

212

R. Goldbach et al. / Virus Research 92 (2003) 207 /212 Schillberg, S., Zimmermann, S., Findlay, K., Fischer, R., 2000. Plasma membrane display of anti-viral single chain Fv fragments confers resistance to tobacco mosaic virus. Mol. Breeding 6 (3), 317 /326. Schillberg, S., Zimmermann, S., Zhang, M.Y., Fischer, R., 2001. Antibody-based resistance to plant pathogens. Transgenic Res. 10 (1), 1 /12. Schouten, A., Roosien, J., Van Engelen, F.A., De Jong, G.A.M., Borst-Vrenssen, A.W.M., Zilverentant, J.F.; Bosch, D., Stiekema, W.J., Gommers, F., 1996. The C-terminal KDEL sequence increases the expression level of a single chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco. Plant Mol. Biol. 30, 781 /793. Spassova, M.I., Prins, T.W., Folkertsma, R.T., Klein-Lankhorst, R.M., Hille, J., Goldbach, R.W., Prins, M., 2001. The tomato gene Sw -5 is a member of the coiled coil, nucleotide binding, leucine-rich repeat class of plant resistance genes and confers resistance to TSWV in tobacco. Mol. Breed. 7 (2), 151 /161. Tavladoraki, P., Benvenuto, E., Trinca S., De Martinis, D., Cattaneo, A., Galef, P., 1993. Transgenic plants expressing a functional single-chain Fv antibody are specically protected from virus attack. Nature 366, 469 /472. Truve, E., Aaspollu, A., Honkanen, J., Puska, R., Mehto, M., Hassi, A., Terri, T.H., Kelve, M., Seppanen, P., Saarma, M., 1993. Transgenic potato plants expressing mammalian 2?,5?-oligoadenylate synthetase are protected from potato virus-x infection under eld conditions. Biotechnology 11 (9), 1048 /1052. Truve, E., Kelve, M., Aaspollu, A., Kuusksalu, A., Seppanen, P., Saarma, M., 1994. Principles and background for the construction of transgenic plants displaying multiple virus-resistance. Arch. Virol. Suppl. (9), 41 /50. Tumer, N.E., Hudak, K., Di, R., Coetzer, C., Wang, P., Zoubenko, O., 1999. Pokeweed antiviral protein and its applications. Curr. Top. Microbiol. Immunol. 240, 139 /158. Van Damme, E.J.M., Hao, Q., Chen, Y., Barre, A., Vandenbussche, F., Desmyter, S., Rouge, P., Peumans, W.J., 2001. Ribosomeinactivating proteins: a family of plant proteins that do more than inactivate ribosomes. Crit. Rev. Plant Sci. 20 (5), 395 /465. Van der Krol, A.R., Mur, L.A., Beld, M., Mol, J.N.M., Stuitje, A., 1990. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2, 291 /299. Voinnet, O., 2001. RNA silencing as a plant immune system against viruses. Trends Genet. 17, 449 /459. Voinnet, O., Pinto, Y.M., Baulcombe, D.C., 1999. Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc. Natl. Acad. Sci. USA 96 (24), 14147 /14152. Voinnet, O., Lederer, C., and Baulcombe, D.C., 2000. A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana Cell 103,157 /167. Wang, P.G., Tumer, N.E., 2000. Virus resistance mediated by ribosome inactivating proteins. Adv. Virus Res. 55, 325 /355. Wang, P.G., Zoubenko, O., Tumer, N.E., 1998. Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II. Plant Mol. Biol. 38 (6), 957 /964. Wilson, T.M.A., 1993. Strategies to protect crop plants against viruses: pathogen-derived resistance blossoms. Proc. Natl. Acad. Sci. USA 90, 3134 /3141. Yelina, N.E., Savenkov, E.I., Solovyev, A.G., Morozov, S.Y., Valkonen, J.P.T., 2002. Long-distance movement, virulence, and RNA silencing suppression controlled by a single protein in hordei- and potyviruses: Complementary functions between virus families. J. Virol., 76 (24), 12981 /12991. Zoubenko, O., Hudak, K., Tumer, N.E., 2000. A non-toxic pokeweed antiviral protein mutant inhibits pathogen infection via a novel salicylic acid-independent pathway. Plant Mol. Biol. 44, 219 /229.

genic plants: exploitation of a cellular pathway possibly involved in RNA degradation. Mol. Plant Microbe Interact. 7, 544 /552. Dunoyer, P., Pfeffer, S., Fritsch, C., Hemmer, O., Voinnet, O., Richards, K.E., 2002. Identication, subcellular localization and some properties of a cysteine-rich suppressor of gene silencing encoded by peanut clump virus. Plant J. 29 (5), 555 /567. Fecker, L.E., Kaufmann, A., Commandeur, U., Commandeur, J., Koenig, R., Burgermeister, W., 1996. Expression of single-chain antibody fragments (scFv) specic for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana. Plant Mol. Biol. 32, 979 /986. Ferreira, S.A., Pitz, K.Y., Manshardt, R., Zee, F., Fitch, M., Gonsalves, D., 2002. Virus coat protein transgenic papaya provides practical control of papaya ringspot virus in Hawaii. Plant Dis. 86 (2), 101 /105. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C., 1998. Potent and specic genetic interference by double-stranded RNA in Caenorhabditis elegans . Nature 391 (6669), 806 /811. Greene, A.E., Allison, R.F., 1994. Recombination between viral RNA and transgenic plant transcripts. Science 263, 1423 /1425. Hull, R., 2002. Matthews Plant Virology. Academic Press, London, p. 1001. Jones, D.A., Jones, J.D.G., 1996. The roles of leucine-rich repeats in plant defences. Adv. Bot. Res. Adv. Plant Pathol. 24, 90 /167. Kajava, A.V., 1998. Structural diversity of leucine-rich repeat proteins. J. Mol. Biol. 277, 519 /527. Kennerdell, J.R., Carthew, R.W., 1998. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 1017 /1026. Lapidot, M., Gafny, R., Ding, B., Wolf, S., Lucas, W.J. and Beachy, R.N., 1993. A dysfunctional movement protein of tobacco mosaic virus that partially modies the plasmodesmata and limits virus spread in transgenic plants. Plant J. 4, 959 /970. Lindbo, J.A., Silva-Rosales, L., Proebsting, W.M., Dougherty, W.G., 1993. Induction of a highly specic antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. Plant Cell 5, 1749 /1759. Lomonossoff, G.P., 1995. Pathogen-derived resistance to plant viruses. Annu. Rev. Phytopathol. 33, 323 /343. Mourrain, P., Beclin, C., Elmayan, T., Feuerbach, F., Godon, C., Morel, J.B., Jouette, D., Lacombe, A.M., Nikic, S., Picault, N., Remoue, K., Sanial, M., Vo, T.A., Vaucheret, H., 2000. Arabidopsis SGS2 and SGS3 genes are required for post-transcriptional gene silencing and natural virus resistance. Cell 101, 533 /542. Napoli, C., Lemieux, C., Jorgensen, R., 1990. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2, 279 / 289. Nielsen, K., Boston, R.S., 2001. Ribosome-inactivating proteins: a plant perspective. Ann. Rev. Plant Phys. Plant Mol. Biol. 52, 785 / 816. Palukaitis, P., Zaitlin, M., 1997. Replicase-mediated resistance to plant virus diseases. Adv. Virus Res. 48, 349 /377. Pfeffer, S., Dunoyer, P., Heim, F., Richards, K. E., Jonard, G., and Ziegler-Graff, V., 2002. P0 of beet Western yellows virus is a suppressor of posttranscriptional gene silencing J. Virol., 76, 6815 / 24. Plasterk, R.H.A., 2002. RNA silencing: The genomes immune system Science 296 (5571), 1263 /1265. Sanford, J.C., Johnson, S.A., 1985. The concept of pathogen-derived resistance. J. Theoret. Biol. 113, 395 /405. Schiebel, W., Pelissier, T., Riedel, L., Thalmeir, S., Schiebel, R., Kempe, D., Lottspeich, F., Sanger, H.L., Wassenegger, M., 1998. Isolation of an RNA-dependent RNA polymerase-specic cDNA clone from tomato. Plant Cell 10, 2087 /2101.