- Email: [email protected]
The dialogue between viruses and hosts in compatible interactions
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The dialogue between viruses and hosts in compatible interactions Andrew Maule*‡, Veronique Leh* and Carsten Lederer*† Understanding the biological principles behind virus-induced symptom expression in plants remains a longstanding challenge. By dissecting the compatible host–virus relationship temporally and genetically, we have begun to map out the relationships of its component parts. The picture that emerges is one in which host gene expression and physiology are under tight temporal control during infection. Addresses *John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK † Cyprus Institute of Neurology and Genetics, International Airport Avenue 6, Agios Dometios, PO Box 23462, Nicosia, Cyprus ‡ e-mail: [email protected]
Figure 1 Viral suppressors of RNAi Viral RNA
dsRNA
RNAi Homology-dependent RNA degradation
Current Opinion in Plant Biology
Systemic signal of RNAi (23 nt dsRNAs)
Current Opinion in Plant Biology 2002, 5: 1369-5266/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1369-5266(02)00272-8 Abbreviations CaMV Cauliflower mosaic virus CMV Cucumber mosaic virus eIF eukaryotic translation initiation factor HSP heat shock protein MP movement protein NADP-ME NADP-dependent malic enzyme PCNA proliferating cell nuclear antigen RNAi RNA interference TMV Tobacco mosaic virus Tom1 Tobamovirus multiplication1
Introduction How virus infection impacts biochemically and physiologically upon cells, tissues and whole plants remains one of the gaps in our knowledge of plant virology. After the first infection event, which occurs after mechanical damage to the cell wall and plasma membrane, the virus remains within the symplast. Further cells are infected only after passage through plasmodesmata. In the absence of an active resistance response (e.g. the hypersensitive response), the cells that have supported a complete infection ‘cycle’ do not die but retain large quantities of virus, while the infection moves to adjacent tissue. Finally, the outcome of the initial infection event and the progressive spread of the virus to most or all susceptible tissue, is the appearance of symptoms. These symptoms represent the sum of physiological and structural changes at the cellular level and alterations in physiology that are associated with the reduced growth and development of the whole plant. Since a cycle of infection may take only a few hours, dissecting the biochemical impact of virus replication at the cellular level is extremely difficult. Nevertheless, a diverse collection of changes has been recorded at the tissue level, some of which have been extrapolated back to cellular events. At the other end of the scale, virus-induced
The accumulation of viruses in plants is regulated by the balance of defence and counter-defence mechanisms whose basis is RNAi. Upon entry into a susceptible cell, viruses initiate a phase of multiplication that requires genome amplification and leads to the accumulation of viral progeny. For most plant viruses, genome amplification requires the participation of a double-stranded RNA (ds RNA) intermediate. This dsRNA is recognised by an endogenous host defence mechanism, which is called post-transcriptional gene silencing or RNAi [1–4,60•]. RNAi leads to the degradation of homologous RNAs in the cytoplasm, so regulating the level of viral RNA accumulation. As a product of this process, small homologous RNAs (of 21–23 nucleotides [nt]) accumulate and are translocated to other tissues of the plant. These molecules may participate in the transmission of a specific signal for RNAi that can lead to the degradation of homologous RNAs at remote sites. Plant viruses have evolved to counter this host defence through the production of suppressors of RNAi. This suppressor activity, which can reside with any one of a diverse range of virus-specific products, blocks RNAi either by disrupting the core mechanism [1–4] or by interfering with the local [61] or distant [62•] translocation of the signal. T-bars indicate inhibition.
biochemical changes in plant protoplast populations have been observed. Whether studying infection at the cellular or tissue level, it has proven difficult to distinguish the virus’ requirements for replication and expression of its genome from factors that are associated with host defence responses or host damage. Placing these events on a temporal scale stretching from early to late events in virus multiplication has also been problematic. A major development in recent years has been the identification of post-transcriptional gene silencing or RNA interference (RNAi) as a host defence response that regulates the level of virus accumulation and influences symptom expression. This process, summarised in Figure 1, has been the subject of many excellent reviews (e.g. [1–4]) and will not be covered further here. Instead, we have focussed on the host’s provision for virus multiplication and the impact that might have on host performance. As a complete multiplication cycle can occur in an individual cell, we look most closely at local requirements and effects, but we also consider the wider impact on symptom expression.
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Cellular factors: positive contributors to virus replication and movement As might be expected, components of the core machineries of the host cell have been implicated in virus replication and the expression of virus genes. However, the extreme intimacy established between the virus and the host means that viable plant mutants that are defective in virus replication have rarely been identified. However, two genes, Tobamovirus multiplication1 (Tom1) and Tom3, were identified from a screen for Arabidopsis thaliana mutants that show defective infection by Tobacco mosaic virus (TMV). Tom1 and Tom3 are putative transmembrane proteins that are proposed to act as membrane anchors for the TMV RNAreplication complex [5,6]. Three candidate factors that appear to be involved in viral replication have emerged from a mutational screen of yeast cells that were engineered to mimic the replication of Brome mosaic virus (BMV) [7]. Viral RNA replication was dependent upon a yeast protein (Lsm1p) that is related to core RNA-splicing factors [8], upon a DEAD-box RNA helicase (DED1) that interfered with BMV RNA2 translation [9], and upon a gene that encodes delta9 fatty acid desaturase (Ole1) [10••]. In the last case, the mutant gene could be complemented by growing the yeast in unsaturated fatty acids, suggesting that replication may not be directly dependent upon the OLE1 protein but is strongly influenced by the fluidity of cellular membranes. This connection with membrane fluidity is consistent with the fact that most plant viruses replicate in association with cellular membranes. Factors that are associated with virus movement seem to be less dependent upon ‘house-keeping’ functions of plant cells than those associated with viral replication. Hence, it has been possible to identify movement-defective plant mutants or variants [11••,12–15]. For the infection to spread to neighbouring cells, viruses need to deregulate the normal constraints on molecular trafficking through plasmodesmata [16]. This is achieved through the action of virus-encoded movement proteins (MPs). In recent years, increasing effort has been aimed at understanding the function of MPs and, in particular, at identifying host targets for their action. There have been some successes. For example, it has been shown that tobamovirus MPs can be phosphorylated in vitro [16,17] and in vivo [16,18] by a host protein kinase, and may interact with pectin methylesterase [21] and/or a transcriptional coactivator [21]. Perhaps most significant in relation to resolving MP function is the requirement for TMV MP to associate with microtubules for TMV cell-to-cell movement to occur [22]. Also, cauliflower mosaic virus (CaMV) MP interacts with an Arabidopsis protein that has homology to mammalian ‘rab acceptor’ [23], and turnip crinkle virus MP interacts with a potential membrane protein from Arabidopsis [24]. Overall, it appears that different virus groups have evolved distinctive ways to modulate cell-to-cell trafficking. Although the impact of these interactions on the host is still unclear, it might be expected that the deregulation of cell-to-cell communication could have profound effects on metabolic
control. This could occur through an imbalance in the trafficking of small molecules or by changes in gene expression mediated by the movement of informational molecules, such as host mRNAs [25]. Intuitively, the former might explain the impact of MP expression on carbohydrate accumulation [26], although recent work with potato leafroll virus MP [27] suggests that the MP determinants of plasmodesmal control and changes in sugars are unlinked. More correlative experimental approaches have identified components of polynucleotide synthesis, and of protein expression and turnover, as necessary components for virus multiplication. For example, the Geminiviridae, which comprise a large group of viruses with DNA genomes, must recruit components of the host’s DNA-synthesising machinery to support their replication in the nuclei of host cells. Some members of the Geminiviridae not only induce the expression and accumulation of proliferating cell nuclear antigen (PCNA; the processivity factor for DNA polymerase delta) [28•] but also interfere with the regulation of the host’s cell cycle (reviewed in [29•]). The latter occurs through the interaction of a virus protein with the retinoblastoma protein, Rb. This interaction can shift mature cells to S phase and create a more favourable cellular environment for active DNA synthesis [29•]. Through studies of the interactions of eukaryotic translation initiation factors with virus proteins or virus RNA, eIF3 was linked to CaMV [30], eIFiso4E and eIF4E to potyviruses [31••,32,33], and heat shock protein 101 (HSP101) to tobamoviruses [34]. Similarly, the CaMV translational transactivator interacts with the L24 [30] and L18 [35] proteins of the 60S ribosomal complex, interactions that are probably important in aiding the unusual polycistronic translation of the CaMV RNA. For a range of viruses (which includes both RNA and DNA viruses) in more than one host, a dramatic but transient increase in the expression of HSP70 and polyubiquitin is associated with infection [36,37•,38••]. This could point separately to chaperone and protein-turnover activity, or could implicate both HSP70 and polyubiquitin in protein targeting to the energydependent proteosome [39]. It is also possible that HSP70 is involved in virion assembly and virus trafficking through plasmodesmata, as a homologous protein that is encoded by closteroviruses has such functions [40,41].
Host responses: from local events to systemic factors To establish clearer relationships between host responses and virus multiplication, experimental approaches have been chosen that take into account the progressive nature of virus infections. Hence, we related time and expanding infection areas in our experiments. We assumed that changes in the host that occur at the advancing ‘infection front’ were immediate or early responses, and those that occur behind the infection front were increasingly late responses. Our collective findings using this approach are summarised in Figure 2. We demonstrated that the induction
The dialogue between viruses and hosts in compatible interactions Maule, Leh and Lederer
The potential for viruses to induce remote and long-range (i.e. systemic) changes in host physiology exists through the deregulation of macromolecular movement from infected cells [25], and through the signalling action of low molecular weight molecules (e.g. hormones, sugars, etc. [26]). A spatial analysis of gene expression in cucurbit tissues infected with Cucumber mosaic virus (CMV) revealed both local and systemic effects [38••]. Expression of HSP70 and NADP-dependent malic enzyme (NADP-ME) was induced in uninfected cells outside of a virus-infected lesion. Notably, NADP-ME expression was increased in an area of tissue that reached approximately 20 cells ahead of the infected area. This gene encodes an anaplerotic enzyme that supplies NADPH for biosynthetic reactions. Although, the mechanism and purpose of induction remain unknown, this increase in NADP-ME activity could prepare the cell for the biosynthetic demands of virus replication. The pattern of NADP-ME induction was distinct from that of the systemic induction of catalase [38••]. In virus-infected peas, Gor2, a gene encoding cytoplasmic glutathione reductase, has an induction pattern that is similar to that of HSP70 [44•]. As oxidative free radicals have the potential to activate defence [47], it is tempting to suggest that the increased generation of glutathione (which acts as an antioxidant) and the activation of catalase (to remove H2O2) represents virus-directed protection against the host defences that operate in compatible interactions. The systemic induction of catalase adds to a list of genes that are similarly induced in a range of compatible host–virus interactions. These include peroxidase, superoxide
Figure 2 Direction of virus invasion (a)
Healthy
Infected Late
Early
(b) Virus replication
Relative expression
of HSP70 and polyubiquitin is immediate, early and transient [36,37•]. We also noted a coordinate response in the depletion of a wide range of host transcripts in recently infected cells [38••,42,43]. This was reminiscent of a commonly observed phenomenon in animal virus infections, termed host-gene ‘shut-off’, and has been widely interpreted as a mechanism to place virus gene expression at a competitive advantage over the expression of host genes. In animal systems shut-off is achieved at diverse points in host gene expression [42]. In the case of Turnip mosaic virus (Potyviridae), it has been proposed that the binding of a viral protein (the turnip mosaic virus virus genome-linked protein [VPg]) to eIFiso4E competitively inhibits the expression of host genes [31••]. In our experiments [38••,43], the simultaneous depletion of many host mRNAs might suggest that active RNA degradation plays a part in suppressing the expression of host genes. The transient nature of the shut-off [42] explains how host cells can continue to function after the virus replication phase is complete. Interestingly, the expression of host genes that encode actin and tubulin remains unchanged during the virus replication phase [44•], presumably reflecting a requirement for the virus to translocate within and between cells using the cytoskeleton [45]. In a different experimental system, no change in actin turnover was observed following virus infection [46].
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(c)
e.g. Actin/ tubulin
(d) Host gene 'shut-off' (e) e.g.HSP70 (f)
Current Opinion in Plant Biology
Catalase NADP-ME (catalase before infection)
Localised changes in the gene expression of the host in response to virus infection of compatible tissues. Changes in the accumulation of host mRNAs are shown for pea tissues that are infected with the Pea seed-borne mosaic virus and Cucurbita pepo tissues that are infected with CMV. (a) The temporal relationship between host responses and an advancing infection front. (b) As might be predicted, virus multiplication is maximal close to the infection but does not persist. (c) The expression of constitutively expressed genes that are required for virus infection (e.g. those encoding actin and tubulin [44•]) remains unchanged. (d) Other mRNAs, which are presumably not required for infection, are depleted (by host gene ‘shut-off’), perhaps to the advantage of the invading virus. This shut-off may be restricted to the area of virus replication (e.g. in pea [42]) or persist for longer periods [38••]. (e,f) Host genes may also be induced in response to virus infection with a range of different induction profiles. (e) Immediate (close to the infection front) and transient induction was seen for HSP70, polyubiquitin and Glutathione reductase2 (Gor2) [36,37•,38••,44•].(f) Systemic induction of catalase [38••] and an intermediate pattern of induction of NADP-ME (for approximately 20 cells beyond the infected area) were seen outside of the infected area in CMV-infected tissues [38••]. These ‘snapshots’ reveal a pattern of host gene expression that is regulated both in time and space with respect to virus infection.
dismutase [48], some pathogenesis-related (PR) genes [12], and glutathione-S-transferase [47]. Some of these genes also contribute to signatures for the hypersensitive response and for systemic acquired resistance, and may indicate the involvement of a general stress response or the invocation of the senescence pathway in response to infection [49]. In these and many other studies of the physiological responses to virus infection in systemically infected tissue, little attention has been given to the precise timing and history of the infection. In the best studies, developmentally equivalent organs were sampled over time. Nevertheless, some common themes emerge from this work. Compatible
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interactions that lead to chlorotic phenotypes are typically associated with a loss of photosynthetic capacity, an increase in respiration, a change in carbohydrate partitioning and altered starch accumulation (e.g. [50]). In a seminal piece of work, Tecsi et al. [51] related CMV-induced physiological changes in cucurbit tissue to the time of virus replication and the timing of symptom expression. By developing technologies for in situ enzyme assays during the development of chlorotic lesions, the order of induced changes relative to the accumulation of virus-specific products was established. In this compatible interaction, CMV replication correlated with a localised increase in photosynthesis, increased NADP-ME activity and increases in the activity of the oxidative pentose phosphate pathway. Potentially, these changes are commensurate with the requirements for increased assimilated carbon and biosynthetic capacity to support virus replication. They were followed by starch accumulation, although this starch was subsequently mobilised. Later after infection, the rates of respiration and glycolysis increased while Benson-Calvin cycle activity increased, and chlorotic symptoms appeared in the centre of the lesion. Hence, it appears that infection sets up a programme of biochemical and physiological changes that lead eventually to symptom expression. This work remains the best-defined biochemical and physiological study of a compatible host–virus interaction.
Reducing complexity: novel investigation strategies We have described a scenario in which a compatible host reacts to virus infection in complex ways that are defined by the demands of the virus, host defences, host stress factors, cellular responses, and local and remote tissue responses. For all of this complexity, we only have a patchwork of more- or less-detailed observations to help us understand the underlying biology. It is clear that further progress will depend on simplified experimental systems and the use of new tools. One approach to studying the host–virus interaction that gives rise to symptoms has been to use the mutagenesis of the virus to define genetic loci and gene products that affect symptom expression (e.g. [52–54]). There have been many recent examples of this approach, but the work has tended to reveal more about the virus than about the biochemistry that underlies symptom production. A second approach has been to engineer the ectopic expression of virus genes in transgenic host plants. This has been more productive in separating the effects of individual gene products from the impact of virus replication. Hence, the expression of the beet curly top virus (a geminivirus) C4 gene has been shown to induce the proliferation of host cells that typifies infection [55]. Constitutive expression of CaMV gene VI, a translational transactivator of the polycistonic viral mRNA, induces severe chlorotic symptoms in transgenic plants that are similar to those seen after CaMV infection [56]. In Arabidopsis, a comparison between
transgenic plants with altered expression of gene VI and CaMV-infected plants identified several genes whose expression patterns were altered in the same way in the two types of plants [57]. Viral movement proteins have also been shown to have a strong impact on the host plant. An MP from Tomato mottle virus (a geminivirus) induced viruslike symptoms when expressed constitutively [58]. Also, the ectopic expression of other MPs has been shown to influence carbohydrate transport and partitioning in the phloem and photosynthesis (reviewed in [26]).
Conclusions Despite several decades of research investigating how viruses influence their susceptible hosts, we remain remarkably ignorant of the factors controlling viral disease. By recognising the progressive nature of the infection process, we have begun to appreciate the complex challenge we face. There are new opportunities to obtain more comprehensive pictures of the effects of virus infection using high-throughput approaches for transcriptome (e.g. microarrays [59]) and metabolome analyses. However, caution needs to be exercised. Certainly a large number of changes will be seen, but their assignment to early/late or direct/indirect effects will not be possible without the development of new ways to control and evaluate the time and location of the infection. The future promise for this research lies in the development and use of synchronous infections, and of inducible systems in plants and cultured cell lines.
Acknowledgements The John Innes Centre is grant-aided by the UK Biotechnology and Biological Research Council (BBSRC). VL was in receipt of an EMBO Research Fellowship. CL was supported through a John Innes Foundation PhD studentship grant.
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48. Riedle-Bauer M: Role of reactive oxygen species and antioxidant enzymes in systemic virus infections of plants. J Phytopathol 2000, 148:297-302. 49. Almasi A, Apatini D, Boka K, Boddi B, Gaborjanyi R: BSMV infection inhibits chlorophyll biosynthesis in barley plants. Physiol Mol Plant Pathol 2000, 56:227-233. 50. Shalatin D, Wolf S: Cucumber mosaic virus infection affects sugar transport in melon plants. Plant Physiol 2000, 123:597-604. 51. Tecsi LE, Smith AM, Maule AJ, Leegood RC: A spatial analysis of physiological changes associated with infection of cotyledons of
marrow plants with cucumber mosaic virus. Plant Physiol 1996, 111:975-985. 52. Morra MR, Petty IT: Tissue specificity of geminivirus infection is genetically determined. Plant Cell 2000, 12:2259-2270. 53. Sáenz P, Cervera MT, Dallot S, Quiot L, Quiot JB, Riechmann JL, Gar′cia JA: Identification of a pathogenicity determinant of Plum pox virus in the sequence encoding the C-terminal region of protein P3+6K(1). J Gen Virol 2000, 81:557-566. 54. Szilassy D, Salánki K, Balázs E: Stunting induced by cucumber mosaic cucumovirus-infected Nicotiana glutinosa is determined by a single amino acid residue in the coat protein. Mol Plant Microbe Interact 1999, 12:1105-1113. 55. Latham JR, Saunders K, Pinner MS, Stanley J: Induction of plant cell division by beet curly top virus gene C4. Plant J 1997, 11:1273-1283. 56. Cecchini EZ, Gong Z, Geri C, Covey SN, Milner JJ: Transgenic Arabidopsis lines expressing gene VI from cauliflower mosaic virus variants exhibit a range of symptom-like phenotypes and accumulate inclusion bodies. Mol Plant Microbe Interact 1997, 10:1094-1101. 57.
Geri C, Cecchini E, Giannakou ME, Covey SN, Milner JJ: Altered patterns of gene expression in Arabidopsis elicited by cauliflower mosaic virus (CaMV) infection and by a CaMV gene VI transgene. Mol Plant Microbe Interact 1999, 12:377-384.
58. Duan YP, Powell CA, Purcifull DE, Broglio P, Hiebert E: Phenotypic variation in transgenic tobacco expressing mutated geminivirus movement/pathogenicity (BC1) proteins. Mol Plant Microbe Interact 1997, 10:1065-1074. 59. Lucas WJ, Wolf S: Connections between virus movement, macromolecular signalling and assimilate allocation. Curr Opin Plant Biol 1999, 2:192-197. 60. Tenllado F, Diaz-Ruíz JR: Double-stranded RNA-mediated • interference with plant virus infection. J Virol 2001, 75:12288-12297. Double-stranded RNA (dsRNA) has been invoked as the trigger for RNAi in plants by extrapolation from other systems and because partially dsRNA hairpin molecules are potent activators of RNAi [1–4]. These authors show that the introduction of dsRNA directly into plant cells generates homologydependent RNAi. 61. Voinnet O, Lederer C, Baulcombe DC: A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 2000, 103:157-167. 62. Guo HS, Ding SW: A viral protein inhibits the long range signaling • activity of the gene silencing signal. EMBO J 2002, 21:398-407. Previously identified viral suppressors of RNAi have targeted the core mechanism [1–4] or the local spread of the silencing signal [61]. For the first time, this work identifies a suppressor that targets the capacity for signalling over long distances, presumably via the phloem.
The dialogue between viruses and hosts in compatible interactions Andrew Maule*‡, Veronique Leh* and Carsten Lederer*† Understanding the biological principles behind virus-induced symptom expression in plants remains a longstanding challenge. By dissecting the compatible host–virus relationship temporally and genetically, we have begun to map out the relationships of its component parts. The picture that emerges is one in which host gene expression and physiology are under tight temporal control during infection. Addresses *John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK † Cyprus Institute of Neurology and Genetics, International Airport Avenue 6, Agios Dometios, PO Box 23462, Nicosia, Cyprus ‡ e-mail: [email protected]
Figure 1 Viral suppressors of RNAi Viral RNA
dsRNA
RNAi Homology-dependent RNA degradation
Current Opinion in Plant Biology
Systemic signal of RNAi (23 nt dsRNAs)
Current Opinion in Plant Biology 2002, 5: 1369-5266/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1369-5266(02)00272-8 Abbreviations CaMV Cauliflower mosaic virus CMV Cucumber mosaic virus eIF eukaryotic translation initiation factor HSP heat shock protein MP movement protein NADP-ME NADP-dependent malic enzyme PCNA proliferating cell nuclear antigen RNAi RNA interference TMV Tobacco mosaic virus Tom1 Tobamovirus multiplication1
Introduction How virus infection impacts biochemically and physiologically upon cells, tissues and whole plants remains one of the gaps in our knowledge of plant virology. After the first infection event, which occurs after mechanical damage to the cell wall and plasma membrane, the virus remains within the symplast. Further cells are infected only after passage through plasmodesmata. In the absence of an active resistance response (e.g. the hypersensitive response), the cells that have supported a complete infection ‘cycle’ do not die but retain large quantities of virus, while the infection moves to adjacent tissue. Finally, the outcome of the initial infection event and the progressive spread of the virus to most or all susceptible tissue, is the appearance of symptoms. These symptoms represent the sum of physiological and structural changes at the cellular level and alterations in physiology that are associated with the reduced growth and development of the whole plant. Since a cycle of infection may take only a few hours, dissecting the biochemical impact of virus replication at the cellular level is extremely difficult. Nevertheless, a diverse collection of changes has been recorded at the tissue level, some of which have been extrapolated back to cellular events. At the other end of the scale, virus-induced
The accumulation of viruses in plants is regulated by the balance of defence and counter-defence mechanisms whose basis is RNAi. Upon entry into a susceptible cell, viruses initiate a phase of multiplication that requires genome amplification and leads to the accumulation of viral progeny. For most plant viruses, genome amplification requires the participation of a double-stranded RNA (ds RNA) intermediate. This dsRNA is recognised by an endogenous host defence mechanism, which is called post-transcriptional gene silencing or RNAi [1–4,60•]. RNAi leads to the degradation of homologous RNAs in the cytoplasm, so regulating the level of viral RNA accumulation. As a product of this process, small homologous RNAs (of 21–23 nucleotides [nt]) accumulate and are translocated to other tissues of the plant. These molecules may participate in the transmission of a specific signal for RNAi that can lead to the degradation of homologous RNAs at remote sites. Plant viruses have evolved to counter this host defence through the production of suppressors of RNAi. This suppressor activity, which can reside with any one of a diverse range of virus-specific products, blocks RNAi either by disrupting the core mechanism [1–4] or by interfering with the local [61] or distant [62•] translocation of the signal. T-bars indicate inhibition.
biochemical changes in plant protoplast populations have been observed. Whether studying infection at the cellular or tissue level, it has proven difficult to distinguish the virus’ requirements for replication and expression of its genome from factors that are associated with host defence responses or host damage. Placing these events on a temporal scale stretching from early to late events in virus multiplication has also been problematic. A major development in recent years has been the identification of post-transcriptional gene silencing or RNA interference (RNAi) as a host defence response that regulates the level of virus accumulation and influences symptom expression. This process, summarised in Figure 1, has been the subject of many excellent reviews (e.g. [1–4]) and will not be covered further here. Instead, we have focussed on the host’s provision for virus multiplication and the impact that might have on host performance. As a complete multiplication cycle can occur in an individual cell, we look most closely at local requirements and effects, but we also consider the wider impact on symptom expression.
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Cellular factors: positive contributors to virus replication and movement As might be expected, components of the core machineries of the host cell have been implicated in virus replication and the expression of virus genes. However, the extreme intimacy established between the virus and the host means that viable plant mutants that are defective in virus replication have rarely been identified. However, two genes, Tobamovirus multiplication1 (Tom1) and Tom3, were identified from a screen for Arabidopsis thaliana mutants that show defective infection by Tobacco mosaic virus (TMV). Tom1 and Tom3 are putative transmembrane proteins that are proposed to act as membrane anchors for the TMV RNAreplication complex [5,6]. Three candidate factors that appear to be involved in viral replication have emerged from a mutational screen of yeast cells that were engineered to mimic the replication of Brome mosaic virus (BMV) [7]. Viral RNA replication was dependent upon a yeast protein (Lsm1p) that is related to core RNA-splicing factors [8], upon a DEAD-box RNA helicase (DED1) that interfered with BMV RNA2 translation [9], and upon a gene that encodes delta9 fatty acid desaturase (Ole1) [10••]. In the last case, the mutant gene could be complemented by growing the yeast in unsaturated fatty acids, suggesting that replication may not be directly dependent upon the OLE1 protein but is strongly influenced by the fluidity of cellular membranes. This connection with membrane fluidity is consistent with the fact that most plant viruses replicate in association with cellular membranes. Factors that are associated with virus movement seem to be less dependent upon ‘house-keeping’ functions of plant cells than those associated with viral replication. Hence, it has been possible to identify movement-defective plant mutants or variants [11••,12–15]. For the infection to spread to neighbouring cells, viruses need to deregulate the normal constraints on molecular trafficking through plasmodesmata [16]. This is achieved through the action of virus-encoded movement proteins (MPs). In recent years, increasing effort has been aimed at understanding the function of MPs and, in particular, at identifying host targets for their action. There have been some successes. For example, it has been shown that tobamovirus MPs can be phosphorylated in vitro [16,17] and in vivo [16,18] by a host protein kinase, and may interact with pectin methylesterase [21] and/or a transcriptional coactivator [21]. Perhaps most significant in relation to resolving MP function is the requirement for TMV MP to associate with microtubules for TMV cell-to-cell movement to occur [22]. Also, cauliflower mosaic virus (CaMV) MP interacts with an Arabidopsis protein that has homology to mammalian ‘rab acceptor’ [23], and turnip crinkle virus MP interacts with a potential membrane protein from Arabidopsis [24]. Overall, it appears that different virus groups have evolved distinctive ways to modulate cell-to-cell trafficking. Although the impact of these interactions on the host is still unclear, it might be expected that the deregulation of cell-to-cell communication could have profound effects on metabolic
control. This could occur through an imbalance in the trafficking of small molecules or by changes in gene expression mediated by the movement of informational molecules, such as host mRNAs [25]. Intuitively, the former might explain the impact of MP expression on carbohydrate accumulation [26], although recent work with potato leafroll virus MP [27] suggests that the MP determinants of plasmodesmal control and changes in sugars are unlinked. More correlative experimental approaches have identified components of polynucleotide synthesis, and of protein expression and turnover, as necessary components for virus multiplication. For example, the Geminiviridae, which comprise a large group of viruses with DNA genomes, must recruit components of the host’s DNA-synthesising machinery to support their replication in the nuclei of host cells. Some members of the Geminiviridae not only induce the expression and accumulation of proliferating cell nuclear antigen (PCNA; the processivity factor for DNA polymerase delta) [28•] but also interfere with the regulation of the host’s cell cycle (reviewed in [29•]). The latter occurs through the interaction of a virus protein with the retinoblastoma protein, Rb. This interaction can shift mature cells to S phase and create a more favourable cellular environment for active DNA synthesis [29•]. Through studies of the interactions of eukaryotic translation initiation factors with virus proteins or virus RNA, eIF3 was linked to CaMV [30], eIFiso4E and eIF4E to potyviruses [31••,32,33], and heat shock protein 101 (HSP101) to tobamoviruses [34]. Similarly, the CaMV translational transactivator interacts with the L24 [30] and L18 [35] proteins of the 60S ribosomal complex, interactions that are probably important in aiding the unusual polycistronic translation of the CaMV RNA. For a range of viruses (which includes both RNA and DNA viruses) in more than one host, a dramatic but transient increase in the expression of HSP70 and polyubiquitin is associated with infection [36,37•,38••]. This could point separately to chaperone and protein-turnover activity, or could implicate both HSP70 and polyubiquitin in protein targeting to the energydependent proteosome [39]. It is also possible that HSP70 is involved in virion assembly and virus trafficking through plasmodesmata, as a homologous protein that is encoded by closteroviruses has such functions [40,41].
Host responses: from local events to systemic factors To establish clearer relationships between host responses and virus multiplication, experimental approaches have been chosen that take into account the progressive nature of virus infections. Hence, we related time and expanding infection areas in our experiments. We assumed that changes in the host that occur at the advancing ‘infection front’ were immediate or early responses, and those that occur behind the infection front were increasingly late responses. Our collective findings using this approach are summarised in Figure 2. We demonstrated that the induction
The dialogue between viruses and hosts in compatible interactions Maule, Leh and Lederer
The potential for viruses to induce remote and long-range (i.e. systemic) changes in host physiology exists through the deregulation of macromolecular movement from infected cells [25], and through the signalling action of low molecular weight molecules (e.g. hormones, sugars, etc. [26]). A spatial analysis of gene expression in cucurbit tissues infected with Cucumber mosaic virus (CMV) revealed both local and systemic effects [38••]. Expression of HSP70 and NADP-dependent malic enzyme (NADP-ME) was induced in uninfected cells outside of a virus-infected lesion. Notably, NADP-ME expression was increased in an area of tissue that reached approximately 20 cells ahead of the infected area. This gene encodes an anaplerotic enzyme that supplies NADPH for biosynthetic reactions. Although, the mechanism and purpose of induction remain unknown, this increase in NADP-ME activity could prepare the cell for the biosynthetic demands of virus replication. The pattern of NADP-ME induction was distinct from that of the systemic induction of catalase [38••]. In virus-infected peas, Gor2, a gene encoding cytoplasmic glutathione reductase, has an induction pattern that is similar to that of HSP70 [44•]. As oxidative free radicals have the potential to activate defence [47], it is tempting to suggest that the increased generation of glutathione (which acts as an antioxidant) and the activation of catalase (to remove H2O2) represents virus-directed protection against the host defences that operate in compatible interactions. The systemic induction of catalase adds to a list of genes that are similarly induced in a range of compatible host–virus interactions. These include peroxidase, superoxide
Figure 2 Direction of virus invasion (a)
Healthy
Infected Late
Early
(b) Virus replication
Relative expression
of HSP70 and polyubiquitin is immediate, early and transient [36,37•]. We also noted a coordinate response in the depletion of a wide range of host transcripts in recently infected cells [38••,42,43]. This was reminiscent of a commonly observed phenomenon in animal virus infections, termed host-gene ‘shut-off’, and has been widely interpreted as a mechanism to place virus gene expression at a competitive advantage over the expression of host genes. In animal systems shut-off is achieved at diverse points in host gene expression [42]. In the case of Turnip mosaic virus (Potyviridae), it has been proposed that the binding of a viral protein (the turnip mosaic virus virus genome-linked protein [VPg]) to eIFiso4E competitively inhibits the expression of host genes [31••]. In our experiments [38••,43], the simultaneous depletion of many host mRNAs might suggest that active RNA degradation plays a part in suppressing the expression of host genes. The transient nature of the shut-off [42] explains how host cells can continue to function after the virus replication phase is complete. Interestingly, the expression of host genes that encode actin and tubulin remains unchanged during the virus replication phase [44•], presumably reflecting a requirement for the virus to translocate within and between cells using the cytoskeleton [45]. In a different experimental system, no change in actin turnover was observed following virus infection [46].
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(c)
e.g. Actin/ tubulin
(d) Host gene 'shut-off' (e) e.g.HSP70 (f)
Current Opinion in Plant Biology
Catalase NADP-ME (catalase before infection)
Localised changes in the gene expression of the host in response to virus infection of compatible tissues. Changes in the accumulation of host mRNAs are shown for pea tissues that are infected with the Pea seed-borne mosaic virus and Cucurbita pepo tissues that are infected with CMV. (a) The temporal relationship between host responses and an advancing infection front. (b) As might be predicted, virus multiplication is maximal close to the infection but does not persist. (c) The expression of constitutively expressed genes that are required for virus infection (e.g. those encoding actin and tubulin [44•]) remains unchanged. (d) Other mRNAs, which are presumably not required for infection, are depleted (by host gene ‘shut-off’), perhaps to the advantage of the invading virus. This shut-off may be restricted to the area of virus replication (e.g. in pea [42]) or persist for longer periods [38••]. (e,f) Host genes may also be induced in response to virus infection with a range of different induction profiles. (e) Immediate (close to the infection front) and transient induction was seen for HSP70, polyubiquitin and Glutathione reductase2 (Gor2) [36,37•,38••,44•].(f) Systemic induction of catalase [38••] and an intermediate pattern of induction of NADP-ME (for approximately 20 cells beyond the infected area) were seen outside of the infected area in CMV-infected tissues [38••]. These ‘snapshots’ reveal a pattern of host gene expression that is regulated both in time and space with respect to virus infection.
dismutase [48], some pathogenesis-related (PR) genes [12], and glutathione-S-transferase [47]. Some of these genes also contribute to signatures for the hypersensitive response and for systemic acquired resistance, and may indicate the involvement of a general stress response or the invocation of the senescence pathway in response to infection [49]. In these and many other studies of the physiological responses to virus infection in systemically infected tissue, little attention has been given to the precise timing and history of the infection. In the best studies, developmentally equivalent organs were sampled over time. Nevertheless, some common themes emerge from this work. Compatible
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interactions that lead to chlorotic phenotypes are typically associated with a loss of photosynthetic capacity, an increase in respiration, a change in carbohydrate partitioning and altered starch accumulation (e.g. [50]). In a seminal piece of work, Tecsi et al. [51] related CMV-induced physiological changes in cucurbit tissue to the time of virus replication and the timing of symptom expression. By developing technologies for in situ enzyme assays during the development of chlorotic lesions, the order of induced changes relative to the accumulation of virus-specific products was established. In this compatible interaction, CMV replication correlated with a localised increase in photosynthesis, increased NADP-ME activity and increases in the activity of the oxidative pentose phosphate pathway. Potentially, these changes are commensurate with the requirements for increased assimilated carbon and biosynthetic capacity to support virus replication. They were followed by starch accumulation, although this starch was subsequently mobilised. Later after infection, the rates of respiration and glycolysis increased while Benson-Calvin cycle activity increased, and chlorotic symptoms appeared in the centre of the lesion. Hence, it appears that infection sets up a programme of biochemical and physiological changes that lead eventually to symptom expression. This work remains the best-defined biochemical and physiological study of a compatible host–virus interaction.
Reducing complexity: novel investigation strategies We have described a scenario in which a compatible host reacts to virus infection in complex ways that are defined by the demands of the virus, host defences, host stress factors, cellular responses, and local and remote tissue responses. For all of this complexity, we only have a patchwork of more- or less-detailed observations to help us understand the underlying biology. It is clear that further progress will depend on simplified experimental systems and the use of new tools. One approach to studying the host–virus interaction that gives rise to symptoms has been to use the mutagenesis of the virus to define genetic loci and gene products that affect symptom expression (e.g. [52–54]). There have been many recent examples of this approach, but the work has tended to reveal more about the virus than about the biochemistry that underlies symptom production. A second approach has been to engineer the ectopic expression of virus genes in transgenic host plants. This has been more productive in separating the effects of individual gene products from the impact of virus replication. Hence, the expression of the beet curly top virus (a geminivirus) C4 gene has been shown to induce the proliferation of host cells that typifies infection [55]. Constitutive expression of CaMV gene VI, a translational transactivator of the polycistonic viral mRNA, induces severe chlorotic symptoms in transgenic plants that are similar to those seen after CaMV infection [56]. In Arabidopsis, a comparison between
transgenic plants with altered expression of gene VI and CaMV-infected plants identified several genes whose expression patterns were altered in the same way in the two types of plants [57]. Viral movement proteins have also been shown to have a strong impact on the host plant. An MP from Tomato mottle virus (a geminivirus) induced viruslike symptoms when expressed constitutively [58]. Also, the ectopic expression of other MPs has been shown to influence carbohydrate transport and partitioning in the phloem and photosynthesis (reviewed in [26]).
Conclusions Despite several decades of research investigating how viruses influence their susceptible hosts, we remain remarkably ignorant of the factors controlling viral disease. By recognising the progressive nature of the infection process, we have begun to appreciate the complex challenge we face. There are new opportunities to obtain more comprehensive pictures of the effects of virus infection using high-throughput approaches for transcriptome (e.g. microarrays [59]) and metabolome analyses. However, caution needs to be exercised. Certainly a large number of changes will be seen, but their assignment to early/late or direct/indirect effects will not be possible without the development of new ways to control and evaluate the time and location of the infection. The future promise for this research lies in the development and use of synchronous infections, and of inducible systems in plants and cultured cell lines.
Acknowledgements The John Innes Centre is grant-aided by the UK Biotechnology and Biological Research Council (BBSRC). VL was in receipt of an EMBO Research Fellowship. CL was supported through a John Innes Foundation PhD studentship grant.
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23. Huang Z, Andrianov VM, Han Y, Howell SH: Identification of Arabidopsis proteins that interact with the cauliflower mosaic virus (CaMV) movement protein. Plant Mol Biol 2001, 47:663-675. 24. Lin B, Heaton LA: An Arabidopsis thaliana protein interacts with a movement protein of Turnip crinkle virus in yeast cells and in vitro. J Gen Virol 2001, 82:1245-1251. 25. Ueki S, Citovsky V: RNA commutes to work: regulation of plant gene expression by systemically transported RNA molecules. Bioessays 2001, 23:1087-1090. 26. Lucas WJ, Wolf S: Connections between virus movement, macromolecular signalling and assimilate allocation. Curr Opin Plant Biol 1999, 2:192-197. 27.
Hofius D, Herbers K, Melzer M, Omid A, Tacke E, Wolf S, Sonnewald U: Evidence for expression level-dependent modulation of carbohydrate status and viral resistance by the potato leafroll virus movement protein in transgenic tobacco plants. Plant J 2001, 28:529-543.
28. Egelkrout EM, Robertson D, Hanley-Bowdoin L: Proliferating cell • nuclear antigen transcription is repressed through an E2F consensus element and activated by geminivirus infection in mature leaves. Plant Cell 2001, 13:1437-1452. Previous work had demonstrated that the geminiviruses stimulate the cell to support viral DNA synthesis through the induced accumulation of PCNA in mature tissues. This work shows that this induction occurs at the level of host gene transcription, most likely by overcoming the repression of the PCNA promoter that is mediated through an E2F consensus element. 29. Gutierrez C: DNA replication and cell cycle in plants: learning from • geminiviruses. EMBO J 2000, 19:792-799. This article reviews an exciting area of research in plant virology, namely the potential of geminiviruses to reverse the fate of fully differentiated plant cells into the S phase of the cell cycle. This phenomenon draws parallels between some DNA viruses in animals and plants, and has the potential to provide elegant tools for the dissection of the plant cell cycle. 30. Park HS, Himmelbach A, Browning KS, Hohn T, Ryabova LA: A plant viral ‘reinitiation’ factor interacts with the host translation machinery. Cell 2001, 106:723-733. 31. Leonard SD, Plante D, Wittmann S, Daigneault N, Fortin MG, •• Lalibertè JF: Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity. J Virol 2000, 74:7730-7737. This paper reports how an interaction between turnip mosaic potyvirus virus genome-linked protein (VPg) and the translation factor eIF(iso)4E may influence virus infectivity. A point mutation in the VPg interfered with the interaction and was associated with reduced symptom formation. Perhaps more significant was the potential for VPg and cellular mRNAs to compete for eIF(iso)4E, so providing the first possible mechanism for the virusinduced shut-off of host plant gene expression [35]. 32. Wittman S, Chatel H, Fortin MG, Laliberte JF: Interaction of the viral protein linked genome of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system. Virology 1997, 21:84-92. 33. Schaad MC, Anderberg RJ, Carrington JC: Strain-specific interaction of the tobacco etch virus NIa protein with the translation initiation factor eIF4E in the yeast two-hybrid system. Virology 2000, 273:300-306. 34. Wells DR, Tanguay RL, Le H, Gallie DR: HSP101 functions as a specific translational regulatory protein whose activity is regulated by nutrient status. Genes Dev 1998, 12:3236-3251. 35. Leh V, Yot P, Keller M: The cauliflower mosaic virus translational transactivator interacts with the 60S ribosomal subunit protein L18 of Arabidopsis thaliana. Virology 2000, 266:1-7. 36. Aranda MA, Escaler M, Wang D, Maule AJ: Induction of HSP70 and polyubiquitin expression associated with plant virus replication. Proc Natl Acad Sci USA 1996, 93:15289-15293. 37. •
Escaler M, Aranda MA, Thomas CL, Maule AJ: Pea embryonic tissues show common responses to the replication of a wide range of viruses. Virology 2000, 267:318-325. Following the initial observations that very localised changes in host gene expression occur in response to potyvirus infection [36,43], this work establishes that the induction of HSP70 and the ‘shut-off’ of other host genes are common responses to a diverse range of RNA and DNA viruses.
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Biotic interactions
38. Havelda Z, Maule AJ: Complex spatial responses to cucumber •• mosaic virus infection in susceptible Cucurbita pepo cotyledons. Plant Cell 2000, 12:1975-1986. This work complements previous biochemical and physiological studies using the same host–virus system [51]. It shows that many of the changes in enzyme activities have parallel changes in host gene expression, emphasising the tight spatial and temporal regulation of host responses following infection. 39. Demand J, Alberti S, Patterson C, Höhfeld J: Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteosome coupling. Curr Biol 2001, 11:1569-1577. 40. Peremyslov VV, Hagiwara Y, Dolja VV: HSP70 homolog functions in cell-to-cell movement of a plant virus. Proc Natl Acad Sci USA 1999, 96:14771-14776. 41. Satyanarayana T, Gowda S, Mawassi M, Albiach-Märtí MR, Ayllón MA, Robertson C, Garnsey SM, Dawson WO: Closterovirus encoded HSP70 homolog and p61 in addition to both coat proteins function in efficient virion assembly. Virology 2000, 278:253-265. 42. Aranda MA, Maule AJ: Virus-induced host gene shutoff in animals and plants. Virology 1998, 243:261-267. 43. Wang D, Maule AJ: Inhibition of host gene expression associated with plant virus replication. Science 1995, 267:229-231. 44. Escaler M, Aranda MA, Roberts IM, Thomas CL, Maule AJ: • A comparison between virus replication and abiotic stress (heat) as modifiers of host gene expression. Mol Plant Pathol 2000, 1:159-167. Recognising the apparent overlap in the nature of plant responses to heat and infection with a Potyvirus, the authors report direct comparisons between responses to the two inducers. Despite the similarities, it appears that virus infection induces a unique range of responses. 45. Aaziz R, Dinant S, Epel BL: Plasmodesmata and plant cytoskeleton. Trends Plant Sci 2001, 6:326-330. 46. Dantán-Gonzalez E, Rosenstein Y, Quinto C, Sánchez F: Actin monoubiquitylation is induced in plants in response to pathogens and symbionts. Mol Plant Microbe Interact 2001, 14:1267-1273. 47.
Gullner G, Tobias I, Fodor J, Komives T: Elevation of glutathione level and activation of glutathione-related enzymes affect virus infection in tobacco. Free Radic Res 1999, 31:S155-S161.
48. Riedle-Bauer M: Role of reactive oxygen species and antioxidant enzymes in systemic virus infections of plants. J Phytopathol 2000, 148:297-302. 49. Almasi A, Apatini D, Boka K, Boddi B, Gaborjanyi R: BSMV infection inhibits chlorophyll biosynthesis in barley plants. Physiol Mol Plant Pathol 2000, 56:227-233. 50. Shalatin D, Wolf S: Cucumber mosaic virus infection affects sugar transport in melon plants. Plant Physiol 2000, 123:597-604. 51. Tecsi LE, Smith AM, Maule AJ, Leegood RC: A spatial analysis of physiological changes associated with infection of cotyledons of
marrow plants with cucumber mosaic virus. Plant Physiol 1996, 111:975-985. 52. Morra MR, Petty IT: Tissue specificity of geminivirus infection is genetically determined. Plant Cell 2000, 12:2259-2270. 53. Sáenz P, Cervera MT, Dallot S, Quiot L, Quiot JB, Riechmann JL, Gar′cia JA: Identification of a pathogenicity determinant of Plum pox virus in the sequence encoding the C-terminal region of protein P3+6K(1). J Gen Virol 2000, 81:557-566. 54. Szilassy D, Salánki K, Balázs E: Stunting induced by cucumber mosaic cucumovirus-infected Nicotiana glutinosa is determined by a single amino acid residue in the coat protein. Mol Plant Microbe Interact 1999, 12:1105-1113. 55. Latham JR, Saunders K, Pinner MS, Stanley J: Induction of plant cell division by beet curly top virus gene C4. Plant J 1997, 11:1273-1283. 56. Cecchini EZ, Gong Z, Geri C, Covey SN, Milner JJ: Transgenic Arabidopsis lines expressing gene VI from cauliflower mosaic virus variants exhibit a range of symptom-like phenotypes and accumulate inclusion bodies. Mol Plant Microbe Interact 1997, 10:1094-1101. 57.
Geri C, Cecchini E, Giannakou ME, Covey SN, Milner JJ: Altered patterns of gene expression in Arabidopsis elicited by cauliflower mosaic virus (CaMV) infection and by a CaMV gene VI transgene. Mol Plant Microbe Interact 1999, 12:377-384.
58. Duan YP, Powell CA, Purcifull DE, Broglio P, Hiebert E: Phenotypic variation in transgenic tobacco expressing mutated geminivirus movement/pathogenicity (BC1) proteins. Mol Plant Microbe Interact 1997, 10:1065-1074. 59. Lucas WJ, Wolf S: Connections between virus movement, macromolecular signalling and assimilate allocation. Curr Opin Plant Biol 1999, 2:192-197. 60. Tenllado F, Diaz-Ruíz JR: Double-stranded RNA-mediated • interference with plant virus infection. J Virol 2001, 75:12288-12297. Double-stranded RNA (dsRNA) has been invoked as the trigger for RNAi in plants by extrapolation from other systems and because partially dsRNA hairpin molecules are potent activators of RNAi [1–4]. These authors show that the introduction of dsRNA directly into plant cells generates homologydependent RNAi. 61. Voinnet O, Lederer C, Baulcombe DC: A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 2000, 103:157-167. 62. Guo HS, Ding SW: A viral protein inhibits the long range signaling • activity of the gene silencing signal. EMBO J 2002, 21:398-407. Previously identified viral suppressors of RNAi have targeted the core mechanism [1–4] or the local spread of the silencing signal [61]. For the first time, this work identifies a suppressor that targets the capacity for signalling over long distances, presumably via the phloem.