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1 Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK
2 Warwick HRI, Wellesbourne, Warwick CV35 9EF, UK
3 Department of Biological Sciences, State University of New York at Buffalo, NY 14260, USA
Correspondence
John P. Carr
jpc1005{at}hermes.cam.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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2b), which cannot express the 2b silencing suppressor protein, cross-protects tobacco (Nicotiana tabacum) and Nicotiana benthamiana plants against disease induction by wild-type Fny-CMV. However, protection is most effective only if inoculation with Fny-CMV2b and challenge inoculation with wild-type CMV occurs on the same leaf. Unexpectedly, Fny-CMV2b also protected plants against infection with TC-CMV, a subgroup II strain that is not closely related to Fny-CMV. Additionally, in situ hybridization revealed that Fny-CMV2b and Fny-CMV can co-exist in the same tissues but these tissues contain zones of Fny-CMV2b-infected host cells from which Fny-CMV appears to be excluded. Taken together, it appears unlikely that cross-protection by Fny-CMV2b occurs by induction of systemic RNA silencing against itself and homologous RNA sequences in wild-type CMV. It is more likely that protection occurs through either induction of very highly localized RNA silencing, or by competition between strains for host cells or resources. Supplementary figures are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Based on serological relationships and sequence criteria, the species is divided into three distinct subgroups: IA, IB and II (Roossinck et al., 1999). CMV has the largest host range of any known plant virus and is transmitted in a non-persistent manner by aphids belonging to over 80 species. Taken together, these factors probably contribute to the worldwide distribution of the virus and its economic importance (Palukaitis et al., 1992; Palukaitis & García-Arenal, 2003). The CMV genome consists of three positive-sense RNA molecules. These are RNAs 1, 2 and 3, which also function as mRNAs for the synthesis of the 1a and 2a replicase proteins, and the 3a movement protein, respectively. During replication, subgenomic mRNAs encoding additional proteins are synthesized. The subgenomic RNA 4, derived from RNA 3, is the mRNA for CMV coat protein (CP) and RNA 4A, which is derived from RNA 2, is the mRNA for the multifunctional 2b protein (Ding et al., 1994).
Direct control of CMV or prevention of its transmission by aphids by using insecticides is difficult to achieve. One promising approach for controlling CMV is the use of pathogen-derived transgenes in genetically modified plants (Palukaitis & Zaitlin, 1997; Gaba et al., 2004). Pathogen-derived resistance to CMV and other viruses works either through the triggering of RNA silencing against the transgene-encoded RNA, or through disruption of one or more stages of viral infection by constitutive expression of wild-type or mutant viral proteins by the host plant (Hellwald & Palukaitis, 1995; Beachy, 1997; Wintermantel & Zaitlin, 2000; Lindbo & Dougherty, 2005). Another means of providing protection through interference with the life cycle of a virus is cross-protection. Cross-protection is a phenomenon in which infection with a mild virus strain protects a plant against infection by closely related, more severe strains of the same virus. Cross-protection was described as early as the 1920s by McKinney (1929) who showed that tobacco plants that had previously been inoculated with a tobacco mosaic virus (TMV) strain causing mild green mosaic symptoms were resistant to a subsequent challenge with a TMV strain that caused yellow mosaic symptoms. Cross-protection has been deployed commercially against a variety of viruses, including TMV and tomato mosaic virus (Rast, 1967a, b), papaya ringspot virus (Yeh & Gonsalves, 1984) and citrus tristeza virus (Costa & Müller, 1980), as well as CMV (Rodriguez-Alvarado et al., 2001).
The mechanism, or mechanisms, behind cross-protection has remained obscure but a number of explanations have been proposed. Currently, the leading hypothesis used to explain cross-protection is that the protective strain induces RNA silencing against its own RNA and homologous sequences, such as those occurring in closely related strains of the same virus (Ratcliff et al., 1999; Hull, 2002; Gal-On & Shiboleth, 2006). Thus, it is hypothesized that the protective strain is acting as an elicitor of a natural antiviral response, RNA silencing, which underlies other natural resistance phenomena, such as recovery and ‘green island’ formation, as well as many instances of pathogen-derived resistance in transgenic plants (Ratcliff et al., 1997; Moore et al., 2001; Voinnet, 2001; Goldbach et al., 2003). Other ideas that have been used to explain cross-protection include competition between protective and challenge virus strains for host cells, intracellular replication sites, host translational apparatus and/or other host factors, or inhibitory interactions between the proteins or nucleic acids of the competing viral strains (Hull & Plaskitt, 1970; Palukaitis & Zaitlin, 1984; Sequeira, 1984; Hull, 2002). Mechanisms such as these may explain ‘exclusion’, in which closely related strains of the same virus infect adjacent cells but do not produce mixed infections within the same host cell (Dietrich & Maiss, 2003; Hull & Plaskitt, 1970).
Many, if not most, viruses have adapted to host resistance mediated by RNA silencing by acquiring silencing suppressor proteins that enable them to evade or blunt the effect of this defence mechanism (Voinnet et al., 1999). Viral suppressor proteins target different points of the machinery regulating induction, amplification and maintenance of RNA silencing (Palukaitis & MacFarlane, 2006), for example by interacting with the small interfering (si) RNAs that confer specificity on RNA silencing (Chapman et al., 2004; Lakatos et al., 2004).
CMV encodes a well studied suppressor of RNA silencing, the 2b protein (Brigneti et al., 1998; Guo & Ding, 2002; Lewsey et al., 2007; Mlotshwa et al., 2002; Zhang et al., 2006). The 2b protein can also act as a symptom determinant; it induces disease symptoms by interfering with microRNA-mediated gene regulation (Lewsey et al., 2007; Zhang et al., 2006). However, the severity of the symptoms induced depends upon the CMV strain and the effects on the host plant of environmental and physiological factors (Handford & Carr, 2007; Lewsey et al., 2007; Zhang et al., 2006).
Genetic engineering of viruses may provide a means of designing and generating mild, potentially cross-protective virus strains (discussed by Gal-On & Shiboleth, 2006). We speculated that if RNA silencing is the mechanism behind cross-protection, then a mutant virus lacking the ability to express a silencing suppressor would be a particularly potent cross-protecting agent. This is because the mutant might act as a trigger of silencing against its own RNA and homologous viral RNA sequences, but would lack the means to inhibit or evade the establishment of RNA silencing. In the present study, we investigated the ability of the CMV mutant CMV2b, which is unable to express the 2b silencing suppressor protein, to cross-protect plants against infection with wild-type CMV strains.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and its deletion mutant Fny-CMV2b was reconstituted by mixing in vitro transcription products of full-length cDNA clones encoding RNA 1 (pFny109), RNA 3 (pFny309), and wild-type RNA 2 (pFny209) or a mutant RNA 2 lacking the 2b open reading frame (ORF) (pFny209/M3), as described previously (Rizzo & Palukaitis, 1990; Ryabov et al., 2001; Soards et al., 2002). Equal volumes of transcripts were combined and gently rubbed with a frosted microscope slide onto tobacco leaves at the three-to-five leaf stage. To prepare inoculum for subsequent experiments, virions of CMV were extracted from the infected plant tissue according to the method of Roossinck & White (1998). A naturally occurring UK isolate of CMV, TC-CMV (N. J. Spence & A. Baker, unpublished data), was maintained in plants of zucchini squash (courgette) (Cucurbita pepo cv. ‘Goldrush’) and propagated for virion purification in tobacco plants. RNA 2 of TC-CMV has been sequenced and has the GenBank accession number EF640931. Experiments with TMV were carried out using xanthi, rather than xanthi-nc, which is resistant to this virus. The naturally occurring TMV mutant YSI/1 was used since it induces clearly observable yellow mosaic symptoms (Banerjee et al., 1995).
Inoculation of plants and RNA extraction.
For cross-protection experiments, tobacco and N. benthamiana plants at the three-to-four leaf stage were inoculated with Fny-CMV2b virions suspended in water at a concentration of 100 µg ml–1. After a period of 9–18 days, the plants were challenge by inoculation with Fny-CMV or TC-CMV at either 1 or 10 µg ml–1. Samples for nucleic acid extraction were harvested from inoculated leaves as well as non-inoculated leaves immediately above the inoculated leaves, or from the uppermost non-inoculated leaves at various times between 17 and 25 days after the challenge inoculation. Nucleic acid was extracted using TRIzol reagent (Invitrogen), DNA was degraded using RQ1 RNase-free DNase (Promega), and the RNA further purified using a Qiagen RNeasy Mini kit, according to the various manufacturers' instructions.
Detection of Fny-CMV and Fny-CMV2b.
RT-PCR was used to detect, and distinguish between, the RNAs 2 of wild-type Fny-CMV and Fny-CMV2b occurring in plant RNA samples. The primers were designed to amplify a region of RNA 2 sequence (nt 2367–3031) flanking the 2b ORF of wild-type Fny-CMV as well as the corresponding region in the RNA 2 of Fny-CMV2b containing a deletion in the 2b ORF (Ryabov et al., 2001) (Fig. 1a). Reverse transcription was carried out using the reverse primer (5'-CCACAAAAGTGGGGGGCACCCG-3') followed by PCR using the reverse and forward primers (5'-AGTACAGAGTTCAGGGTTGAGCGTG-3'). PCR reaction conditions were as follows: 94 °C 5 min, 94 °C 30 s, 65 °C 30 s, 72 °C 1 min 30 s for 30 cycles, final extension at 72 °C for 7 min. PCR products were analysed on 1 % (w/v) agarose gels. In some experiments, the presence of CMV (Fny-CMV or Fny-CMV2b) was detected in leaf tissue by using a rapid immunodiagnostic test kit (Pocket Diagnostics).
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2b on opposite surfaces of the leaf or on adjacent areas on the same surface. At 3–4 weeks after inoculation these co-inoculation zones were excised from leaves and prepared for in situ hybridization by using a protocol adapted from previously published methods (Long & Berry, 1996; Patel et al., 2004; Ruzin, 1999; Wang et al., 1993). Tissue samples were fixed by vacuum infiltration in 1 % (v/v) glutaraldehyde in 0.05 M sodium cacodylate buffer and incubated overnight at 4 °C, then dehydrated through an ethanol/tert-butyl alcohol series and embedded in Paraplast Plus (Monoject Scientific). In some cases, paraformaldehyde was used for fixation (Jackson, 1992). A microtome was used to prepare 5 µm transverse sections through embedded leaf tissues that were transferred onto ‘Superfrost Ultra plus’ slides (VWR) and dried overnight at 42 °C. Slides were deparaffinized by two 15 min incubation steps in xylene. Sections were rehydrated through an ethanol series consisting of 5 min sequential incubations in absolute ethanol (twice), 95, 80, 70, 50 and 30 % (v/v) ethanol. Slides were washed twice for 2 min with sterile, RNase-free water. After exposure to 0.2 M HCl for 20 min at room temperature and incubation in 2x saline sodium citrate (SSC) at 70 °C for 30 min, sections were further incubated with 1 µg proteinase K ml–1 for 30 min at 37 °C and then blocked by 2 mg glycine ml–1 in PBS. Sections were fixed in 10 % (v/v) formalin, washed twice for 5 min in PBS and incubated for 10 min in 0.1 M triethanolamine buffer (pH 8.0) containing 0.5 % (v/v) acetic anhydride. The slides were washed twice for 5 min in PBS and dehydrated through an ethanol series (5 min for each sequential step): 30, 50, 70, 80, 95 % (v/v) and absolute ethanol (two incubations). Finally, the slides were dried and immediately used for pre-hybridization. Slides were incubated for 10 min in 2x SSC before pre-hybridization and hybridization at 50 °C, followed by washing in various dilutions of SSC as described previously (Long & Berry, 1996; Patel et al., 2004; Wang et al., 1993).
Three biotin-labelled riboprobes were synthesized for use in hybridization. The first riboprobe was complementary to the conserved 3'-terminal region of all of the Fny-CMV RNAs. This riboprobe, which has been described previously (Carr et al., 1994; Gal-On et al., 1994), can act as a ‘general’ probe capable of detecting the presence of both wild-type Fny-CMV RNAs and the mutant CMV RNA 2 component of CMV2b. The second riboprobe was designed to bind specifically to a region of sequence within the 2b ORF to detect the presence of wild-type RNA 2 but not the mutant RNA 2 of CMV2b, which contains a deletion in this region (Ryabov et al., 2001; Soards et al., 2002). This probe was synthesized by in vitro transcription of a DNA template generated by PCR from wild-type Fny-CMV RNA 2 cDNA sequence using the primers CMV2B.F (5'-GAACGAGGTCACAAAAGTCC-3') and CMV2.R_T7 (5'-TAATACGACTCACTATAGGGAGACGTCAAAATCATGGTCTTCC-3'). The use of primer CMV2.R_T7 introduces a T7 RNA polymerase promoter sequence into the transcription template. The third riboprobe acted as a negative control and was complementary to an approximately 300 bp long green fluorescent protein (gfp) sequence. Primers GFP-F3 (5'-CGTGCTGAAGTCAAGTT-3') and GFP-R3_T7 (5'-TAATACGACTCACTATAGGGAGACGAAAGGGCAGATTGT-3') were used to amplify a fragment from pF : GFP/CP (Canto et al., 1997) introducing the T7 promoter sequence for in vitro transcription of antisense RNA. The riboprobes were labelled with biotin by including biotin-16-uridine-5'-triphosphate in the transcription reactions, according to the instructions of the manufacturer (Roche Applied Sciences). Biotin-labelled riboprobe binding to tissue sections was detected by using streptavidin-alkaline phosphatase conjugate (NeutrAvidin; Pierce Biotechnology) and the Sigma Fast 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium alkaline phosphate substrate. Developed slides were imaged by using a Nikon ECLIPSE 50i microscope and images were recorded by using a digital camera control unit (Nikon).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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2b protects against a challenge infection with wild-type Fny-CMV2b infections are symptomless, whereas the wild-type subgroup IA strain Fny-CMV induces strong symptoms such as leaf distortion and severe stunting of whole plants (Figs 2 and 3 and Soards et al., 2002). Plants that had previously been inoculated with Fny-CMV2b remained symptomless even when they were later challenged with the wild-type virus on the same leaf. In contrast, plants that had not been pre-inoculated with Fny-CMV2b showed typical Fny-CMV-induced symptoms of stunting and distortion of the leaves (Figs 2 and 3). Pre-inoculation with Fny-CMV2b protected plants against Fny-CMV challenge inocula at concentrations of, at least, up to 10 µg ml–1 (Supplementary Fig. S1 available in JGV Online). Experiments in which plants were infected with Fny-CMV2b and challenged with Fny-CMV were carried out independently six times comprising more than 108 plants in total, and out of 60 Fny-CMV2b-infected plants challenged with Fny-CMV none exhibited stunting or any other disease symptoms characteristic of infection by the wild-type virus (Table 1).
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2b triggers a general plant defence mechanism against all viruses, plants were challenged with an unrelated virus. TMV strain YSI/1 is a yellowing strain of TMV that induces strong, easily observable symptoms on tobacco (Banerjee et al., 1995). As expected, Fny-CMV2b did not protect plants against challenge infection with TMV YSI/1 (Fig. 3
). To explore the broadness of protection provided by Fny-CMV2b, we tested whether it could protect against disease induction by TC-CMV, a CMV subgroup II strain that has an RNA 2 sequence with 70 % similarity to the subgroup IA strain Fny-CMV (unpublished data). Surprisingly, in three independent experiments (24 plants) Fny-CMV2b was able to protect against challenge with TC-CMV (Fig. 2 and data not shown).
To detect the presence of the protective mutant and challenge wild-type viruses in infected plants, RT-PCR was used to analyse total RNA extracted from upper non-inoculated leaves (Fig. 1b–c). In all plants from four separate experiments that had been inoculated with Fny-CMV2b and subsequently challenged with wild-type Fny-CMV on the same leaf (total of 22 plants), no accumulation of wild-type viral RNA was detected by RT-PCR in the inoculated leaves, whereas over 75 % of these leaves contained detectable levels of Fny-CMV2b RNA at 21 days post-challenge (Fig. 1c and data not shown). From the analysis of viral RNA occurring in the upper, non-inoculated leaves, over 63 % of plants showed evidence of systemic spread of Fny-CMV2b (Table 1). Interestingly, four plants (7.4 %) in one of the experiments contained low but detectable amounts of challenge strain RNA (Table 1 and examples in Fig. 1c). Nevertheless, these plants, like all others challenged with the wild-type virus, were symptom free and contained detectable levels of RNA 2 of the Fny-CMV2b mutant. Overall, the results indicate that in the majority of cases inoculation of tobacco plants with Fny-CMV2b protects them against the disease symptoms induced by Fny-CMV and against detectable levels of infection with the challenging virus (Table 1). However, it appears that inoculation with Fny-CMV2b cannot completely prevent some host cells becoming infected with the challenge virus and, in a small proportion of challenged plants, giving rise to a low level, asymptomatic infection with the challenge virus.
Cross-protection by Fny-CMV2b also works against wild-type Fny-CMV in N. benthamiana plants
Due in part to defects in its RNA silencing system and the properties of its plasmodesmata the plant N. benthamiana is exceptionally susceptible to a wide range of wild-type viruses as well as mutants that spread poorly in other hosts (Christie & Crawford, 1978; Howard et al., 2004; Murphy et al., 2004; Yang et al., 2004). We were curious to see if this highly susceptible host plant is protected by Fny-CMV2b against challenge with Fny-CMV. Plants were inoculated with Fny-CMV2b at a concentration of 100 µg ml–1 and challenged after a period of 11–15 days on the same leaf with wild-type Fny-CMV at concentration of 10 µg ml–1. Symptoms were observed over a period of 4 weeks following the challenge inoculation. In three independent experiments, 12 plants that had previously been inoculated with Fny-CMV2b were protected against the symptoms of Fny-CMV infection. In contrast, unprotected plants challenged with Fny-CMV showed symptoms of vein clearing, yellowing, leaf distortion and stunting of whole plants after 15 days. The results demonstrate that the protection induced by Fny-CMV2b against Fny-CMV also works in hosts other than tobacco (Fig. 4).
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2b could protect plants from Fny-CMV and TC-CMV, it is not a realistic model for the potential practical application of Fny-CMV2b-mediated cross-protection. Therefore, experiments were conducted in which Fny-CMV2b-inoculated plants were challenged on a different leaf. Tobacco plants were inoculated with Fny-CMV2b and 6 days later tested with an immunodiagnostic test kit to ensure that the inoculation had been successful. Confirmed Fny-CMV2b-infected plants were then inoculated with Fny-CMV on a randomly chosen upper leaf 8 days following the primary inoculation. Symptom development was monitored for 8 weeks following the first inoculation, after which RNA was extracted from the upper non-inoculated leaves of the plants (Fig. 5). Fny-CMV2b-infected N. benthamiana plants were challenge inoculated at 15 days post-inoculation, monitored for symptom development for 6 weeks, at which point samples were taken for RT-PCR analysis (data not shown). In both tobacco and N. benthamiana plants, a total of 16 plants inoculated with Fny-CMV2b and subsequently challenged on the same leaf with Fny-CMV did not develop any local or systemic symptoms at all, consistent with previous results. However, almost 50 % of plants challenged with Fny-CMV on a different leaf (total of 17 plants) were not protected from the induction of visible systemic disease symptoms. RT-PCR analysis showed that the upper leaves of plants displaying symptoms contained wild-type Fny-CMV RNA, whereas equivalent samples from symptomless plants contained Fny-CMV2b RNA (Fig. 5 and data not shown).
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2b did not appear to be caused by induction of systemic RNA silencing it was possible that the mutant was inhibiting infection by the challenging virus by excluding it. That is, by competition for host cells. We predicted that if this idea was correct, co-infected tissue would contain islands of cells infected only by the mutant. It was possible to investigate the distribution of the two viruses in doubly infected tobacco leaf tissue by taking sequential, serial sections through the same piece of inoculated leaf tissue and incubating them with biotin-labelled riboprobes specific to either the 2b sequence of Fny-CMV or the 3'-terminal sequence shared by the RNAs 2 of Fny-CMV and Fny-CMV2b (Fig. 6). In sections of tissue where infection of cells by both viruses had occurred, sectors of tissue were labelled by the 3'-end-specific riboprobe, but not by the probe specific for the 2b gene sequence, while nearby regions of tissue were labelled by both riboprobes (Fig. 6 and Supplementary Fig. S2 for additional controls and examples). This indicated that cells infected with Fny-CMV2b had not become doubly infected with the wild-type virus and the data suggest that cells infected with the mutant resist entry by the wild-type virus. Since RNA sequences belonging to both the wild-type and mutant versions of Fny-CMV were readily detectable in adjacent cells, the data further support the idea that infection with Fny-CMV2b does not induce strong systemic silencing of homologous RNA sequences.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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2b also protected against a less closely related strain, TC-CMV. One of the leading theories put forward to explain cross-protection is that the protective virus strain triggers RNA silencing directed against homologous sequences occurring in the genome of the challenged strain (Kurihara & Watanabe, 2003; Ratcliff et al., 1999; Valkonen et al., 2002). On the face of it, the cross-protection afforded by Fny-CMV2b appears to be consistent with this hypothesis. This is because a virus lacking an RNA silencing suppressor should be a particularly effective cross-protecting strain by virtue of inducing RNA silencing against itself and viral strains possessing homologous sequences.
Previous work has shown that viral mutants compromised in the expression of a silencing suppressor can induce protection against homologous viral sequences. Mutant tombusviruses unable to express the gene for the P19 silencing suppressor protein can infect plants, but the plants subsequently recover from infection due to the degradation of viral RNA mediated by virus-specific siRNAs (Silhavy et al., 2002). Recovered plants show silencing-mediated resistance to viral constructs with similarity to the inducing virus. Thus, plants initially infected with Cym19stop, a mutant of cymbidium ringspot virus (CymRSV), were resistant to infection by potato virus X (PVX)-derived vectors carrying CymRSV sequences (Szittya et al., 2002).
Some of our findings appear to be inconsistent with a model for Fny-CMV2b-mediated cross-protection based on RNA silencing. For example, the ability of the mutant, which is derived from a subgroup IA strain, Fny-CMV2b, to protect against a subgroup II strain was unexpected since they are not highly homologous. Based on alignment of the RNA 2 sequences of the two wild-type viruses, Fny-CMV and TC-CMV share only 70 % sequence similarity overall. Thomas et al. (2001) determined that 23 nt of identity or near identity between sequences was the minimum needed in principle to generate identity-based silencing, but alignment of the two RNA 2 sequences indicates that they have few regions in common with identical sequence exceeding 20 nt (data not shown). In contrast, most conventional examples of cross-protection only work when the strains are more closely related than this (Hull, 2002). Similarly, where plants have been genetically engineered to resist CMV with a virus-derived transgene and the protection results from a combination of RNA-mediated and protein-mediated mechanisms, resistance is only effective against CMV strains belonging to the same subgroup as the virus strain from which the transgene sequence was derived (Carr et al., 1994; Hellwald & Palukaitis, 1995; Wintermantel & Zaitlin, 2000; Zaitlin et al., 1994).
Our results indicate that it is unlikely that infection with Fny-CMV2b results in the generation of a strong systemic silencing signal directed against CMV-specific RNA sequences. It was found that the location of the challenge inoculation site relative to the site inoculated with the protective strain had a clear effect on the degree of protection. Thus, when tobacco and N. benthamiana plants were challenged with Fny-CMV on a leaf different from that inoculated with Fny-CMV2b, almost 50 % of plants displayed symptoms typical of the challenging virus, although the progression of disease in these plants was slowed down by up to a week (Fig. 5 and data not shown).
Since Fny-CMV2b appeared unlikely to be providing protection based on its similarity to sequences in the challenge viruses or by inducing a strong systemic silencing signal, this suggested that one of two potential mechanisms might explain how this form of cross-protection may operate. Firstly, Fny-CMV2b might induce highly localized, non-systemic RNA silencing against homologous sequences in the challenging virus. Highly localized RNA silencing can be induced by viral mutants lacking the gene for a silencing suppressor protein; for example, a P38 (a CP) deletion mutant of tobacco crinkle virus (Ryabov et al., 2004). Highly localized RNA silencing, without detectable accumulation of siRNAs, can also occur in certain lines of transgenic plants overexpressing the gfp reporter gene (Kalantidis et al., 2006). Secondly, it is possible that the presence of the protecting virus may exclude the challenge strain from cells that it has infected by occupying sites within the host cell or titrating out host factors needed by the challenging virus strain.
Mutual exclusion of closely related strains of the same virus has been demonstrated previously. For example, it was found that two genetically modified versions of the same plum pox virus (PPV) strain, one expressing GFP and the other a red fluorescent protein, very infrequently infected the same host cells (Dietrich & Maiss, 2003). In contrast, PPV expressing either of the fluorescent proteins was able to co-infect cells with PVX expressing GFP or the red fluorescent protein (Dietrich & Maiss, 2003). Hull & Plaskitt (1970), using electron microscopy to identify strain-specific ultrastructural features in infected cells, demonstrated a similar exclusion effect in tissues infected with closely related strains of alfalfa mosaic virus. Similarly, using in situ hybridization, Takeshita et al. (2004) found that two strains of CMV did not mix in cells of co-infected cowpea plants. Interestingly, these CMV strains belonged to different CMV subgroups (Takeshita et al., 2004), possibly making it less likely that this exclusion effect resulted from RNA silencing.
Early in infection of tobacco, wild-type CMV and CMV2b move preferentially into different cell types with the mutant moving more rapidly into and through the mesophyll cell layer, which is the predominant cell type in leaves (Soards et al., 2002). To investigate the possibility that Fny-CMV2b excludes wild-type CMV from the cells it infects, we investigated the relative distribution of wild-type Fny-CMV and Fny-CMV2b in doubly infected tissue by using in situ hybridization. Our prediction that we should observe zones of cells infected with Fny-CMV2b immediately adjacent to cells harbouring wild-type viral sequences was substantiated. The fact that we could observe this pattern of viral RNA distribution in simultaneously inoculated areas of tissue is suggestive that exclusion of the unmodified virus by Fny-CMV2b does not require induction of RNA silencing. However, our results cannot exclude a role for highly localized silencing and only further experiments using mutant plants compromised in the silencing machinery (for example, see Deleris et al., 2006) may provide a definitive answer.
Recently, it was described how introduction of specific mutations into the gene for the HC-Pro-silencing suppressor protein of zucchini yellow mosaic virus (ZYMV) yielded mild strains that cross-protected host plants from a severe ZYMV strain (Lin et al., 2007). Despite this, the HC-Pro of the mild ZYMV mutant was still an effective suppressor of RNA silencing (Lin et al., 2007). These results, together with our data on cross-protection afforded by Fny-CMV2b, show that modification or deletion of genes encoding silencing suppressor proteins is a viable method of generating mild or non-symptom-inducing strains for testing as potential cross-protecting agents. However, it may not necessarily follow that the cross-protecting properties of these mutant viruses are due to the induction of RNA silencing against homologous sequences in the challenge virus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Beachy, R. N. (1997). Mechanisms and applications of pathogen-derived resistance in transgenic plants. Curr Opin Biotechnol 8, 215–220.[CrossRef][Medline]
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, 6739–6746.[CrossRef][Medline]
Canto, T., Prior, D. A., Hellwald, K. H., Oparka, K. J. & Palukaitis, P. (1997). Characterization of cucumber mosaic virus. IV. Movement protein and coat protein are both essential for cell-to-cell movement of cucumber mosaic virus. Virology 237, 237–248.[CrossRef][Medline]
Carr, J. P., Gal-On, A., Palukaitis, P. & Zaitlin, M. (1994). Replicase-mediated resistance to cucumber mosaic virus in transgenic plants involves suppression of both virus replication in the inoculated leaves and long distance movement. Virology 199, 439–447.[CrossRef][Medline]
Chapman, E. J., Prokhnevsky, A. I., Gopinath, K., Dolja, V. V. & Carrington, J. C. (2004). Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev 18, 1179–1186.
Christie, S. R. & Crawford, W. E. (1978). Plant virus range of Nicotiana benthamiana. Plant Dis Rep 62, 20–22.
Costa, A. S. & Müller, G. W. (1980). Tristeza control by cross protection: a US-Brazil cooperative success. Plant Dis 64, 538–541.
Deleris, A., Gallego-Bartolome, J., Bao, J., Kasschau, K. D., Carrington, J. C. & Voinnet, O. (2006). Hierarchical action and inhibition of plant dicer-like proteins in antiviral defense. Science 313, 68–71.
Dietrich, C. & Maiss, E. (2003). Fluorescent labelling reveals spatial separation of potyvirus populations in mixed infected Nicotiana benthamiana plants. J Gen Virol 84, 2871–2876.
Ding, S.-W., Anderson, B. J., Haase, H. R. & Symons, R. H. (1994). New overlapping gene encoded by the cucumber mosaic virus genome. Virology 198, 593–601.[CrossRef][Medline]
Gaba, V., Zelcer, A. & Gal-On, A. (2004). Invited review: cucurbit biotechnology – the importance of virus resistance. In Vitro Cell Dev Biol Plant 40, 346–358.[CrossRef]
Gal-On, A. & Shiboleth, Y. M. (2006). Cross-protection. In Natural Resistance Mechanisms of Plants to Viruses, pp. 261–288. Edited by G. Loebenstein & J. P. Carr. Netherlands: Springer Publishers.
Gal-On, A., Kaplan, I., Roossinck, M. J. & Palukaitis, P. (1994). The kinetics of infection of zucchini squash by cucumber mosaic virus indicate a function for RNA 1 in virus movement. Virology 205, 280–289.[CrossRef][Medline]
Goldbach, R., Bucher, E. & Prins, M. (2003). Resistance mechanisms to plant viruses: an overview. Virus Res 92, 207–212.[CrossRef][Medline]
Guo, H. S. & Ding, S.-W. (2002). A viral protein inhibits the long range signaling activity of the gene silencing signal. EMBO J 21, 398–407.[CrossRef][Medline]
Handford, M. G. & Carr, J. P. (2007). A defect in carbohydrate metabolism ameliorates symptom severity in virus-infected Arabidopsis thaliana. J Gen Virol 88, 337–341.
Hellwald, K. H. & Palukaitis, P. (1995). Viral RNA as a potential target for two independent mechanisms of replicase-mediated resistance against cucumber mosaic virus. Cell 83, 937–946.[CrossRef][Medline]
Howard, A. R., Heppler, M. L., Ju, H.-J., Krishnamurthy, K., Payton, M. E. & Verchot-Lubicz, J. (2004). Potato virus X TGBp1 induces plasmodesmata gating and moves between cells in several host species whereas CP moves only in N. benthamiana leaves. Virology 328, 185–197.[CrossRef][Medline]
Hull, R. (2002). Matthews' Plant Virology, 4th edn. New York: Academic Press.
Hull, R. & Plaskitt, A. (1970). Electron microscopy on the behaviour of two strains of alfalfa mosaic virus in mixed infections. Virology 42, 773–776.[CrossRef][Medline]
Jackson, D. (1992). In situ hybridization in plants. In Molecular Plant Pathology, vol. I, a Practical Approach. Edited by S. J. Gurr, M. J. McPherson & D. J. Bowles. Oxford: Oxford University Press.
Kalantidis, K., Tsagris, M. & Tabler, M. (2006). Spontaneous short-range silencing of a GFP transgene in Nicotiana benthamiana is possibly mediated by small quantities of siRNA that do not trigger systemic silencing. Plant J 45, 1006–1016.[Medline]
Kurihara, Y. & Watanabe, Y. (2003). Cross-protection in Arabidopsis against crucifer tobamovirus Cg by an attenuated strain of the virus. Mol Plant Pathol 4, 259–269.[CrossRef]
Lakatos, L., Szittya, G., Silhavy, D. & Burgyan, J. (2004). Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses. EMBO J 23, 876–884.[CrossRef][Medline]
Lewsey, M., Robertson, F. C., Canto, T., Palukaitis, P. & Carr, J. P. (2007). Selective targeting of miRNA-regulated plant development by a viral counter-silencing protein. Plant J 50, 240–252.[CrossRef][Medline]
Lin, S.-S., Wu, H.-W., Jan, F.-J., Hou, R. F. & Yeh, S.-D. (2007). Modifications of the helper component-protease of Zucchini yellow mosaic virus for generation of attenuated mutants for cross protection against severe infection. Phytopathol 97, 287–296.[CrossRef]
Lindbo, J. A. & Dougherty, W. G. (2005). Plant pathology and RNAi: a brief history. Annu Rev Phytopathol 43, 191–204.[CrossRef][Medline]
Long, J. J. & Berry, J. O. (1996). Tissue-specific and light-mediated expression of the C4 photosynthetic NAD-dependent malic enzyme of amaranth mitochondria. Plant Physiol 112, 473–482.[Abstract]
McKinney, H. H. (1929). Mosaic diseases in the Canary Islands, West Africa and Gibraltar. J Agric Res 39, 557–578.
Mlotshwa, S., Voinnet, O., Mette, M. F., Matzke, M., Vaucheret, H., Ding, S. W., Pruss, G. & Vance, V. B. (2002). RNA silencing and the mobile silencing signal. Plant Cell 14 (Suppl), S289–S301.
Moore, C. J., Sutherland, P. W., Forster, R. L. S., Gardner, R. C. & MacDiarmid, R. M. (2001). Dark green islands in plant virus infection are the result of posttranscriptional gene silencing. Mol Plant Microbe Interact 14, 939–946.[Medline]
Murphy, A. M., Gilliland, A., York, C. J., Hyman, B. & Carr, J. P. (2004). High-level expression of alternative oxidase protein sequences enhances the spread of viral vectors in resistant and susceptible plants. J Gen Virol 85, 3777–3786.
Palukaitis, P. & García-Arenal, F. (2003). Cucumoviruses. Adv Virus Res 62, 241–323.[Medline]
Palukaitis, P. & MacFarlane, S. (2006). Viral counter-defense molecules. In Natural Resistance Mechanisms of Plants to Viruses, pp. 165–185. Edited by G. Loebenstein & J. P. Carr. Netherlands: Springer Publishers.
Palukaitis, P. & Zaitlin, M. (1984). A model to explain the ‘cross-protection’ phenomenon shown by plant viruses and viroids. In Plant–Microbe Interactions, pp. 420–429. Edited by T. Kosuge & E. Nester. New York: Macmillan.
Palukaitis, P. & Zaitlin, M. (1997). Replicase-mediated resistance to plant virus disease. Adv Virus Res 48, 349–377.[Medline]
Palukaitis, P., Roossinck, M. J., Dietzgen, R. G. & Francki, R. I. (1992). Cucumber mosaic virus. Adv Virus Res 41, 281–348.[Medline]
Patel, M., Corey, A. C., Yin, L. P., Ali, S. J., Taylor, W. C. & Berry, J. O. (2004). Untranslated regions from C-4 amaranth AhRbcS1 mRNAs confer translational enhancement and preferential bundle sheath cell expression in transgenic C-4 Flaveria bidentis. Plant Physiol 136, 3550–3561.
Rast, A. T. B. (1967a). Differences in aggressiveness between TMV isolates from tomato on clones of Lycopersicum peruvianum. Eur J Plant Pathol 73, 186–189.
Rast, A. T. B. (1967b). Yield of glasshouse tomatoes as affected by strains of tobacco mosaic virus. Eur J Plant Pathol 73, 147–156.
Ratcliff, F., Harrison, B. D. & Baulcombe, D. C. (1997). A similarity between viral defense and gene silencing in plants. Science 276, 1558–1560.
Ratcliff, F. G., MacFarlane, S. A. & Baulcombe, D. C. (1999). Gene silencing without DNA. RNA-mediated cross-protection between viruses. Plant Cell 11, 1207–1216.
Rizzo, T. M. & Palukaitis, P. (1990). Construction of full-length cDNA clones of cucumber mosaic virus RNAs 1, 2 and 3: generation of infectious RNA transcripts. Mol Gen Genet 222, 249–256.[CrossRef][Medline]
Rodriguez-Alvarado, G., Kurath, G. & Dodds, J. A. (2001). Cross-protection between and within subgroup I and II of cucumber mosaic virus isolates from pepper. Agrociencia 35, 563–573.
Roossinck, M. J. & Palukaitis, P. (1990). Rapid induction and severity of symptoms in zucchini squash (Cucurbita pepo) map to RNA 1 of cucumber mosaic virus. Mol Plant Microbe Interact 3, 188–192.
Roossinck, M. J. & White, P. S. (1998). Cucumovirus isolation and RNA extraction. In Methods in Molecular Biology. Plant Virus Protocols. From Virus Isolation to Transgenic Resistance, pp. 189–196. Edited by G. D. Foster & S. C. Taylor. Totowa, NJ: Humana Press.
Roossinck, M. J., Zhang, L. & Hellwald, K. H. (1999). Rearrangements in the 5' nontranslated region and phylogenetic analyses of cucumber mosaic virus RNA 3 indicate radial evolution of three subgroups. J Virol 73, 6752–6758.
Ruzin, S. E. (1999). Plant Microtechnique and Microscopy. New York: Oxford University Press, Inc.
Ryabov, E. V., Fraser, G., Mayo, M. A., Barker, H. & Taliansky, M. (2001). Umbravirus gene expression helps Potato leafroll virus to invade mesophyll tissues and to be transmitted mechanically between plants. Virology 286, 363–372.[CrossRef][Medline]
Ryabov, E. V., van Wezel, R., Walsh, J. & Hong, Y. (2004). Cell-to-cell, but not long-distance, spread of RNA silencing that is induced in individual epidermal cells. J Virol 78, 3149–3154.
Sequeira, L. (1984). Recognition systems in plant-pathogen interactions. Biol Cell 51, 281–286.
Silhavy, D., Molnár, A., Lucioli, A., Szittya, G., Hornyik, C., Tavazza, M. & Burgyán, J. (2002). A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21, 3070–3080.[CrossRef][Medline]
Soards, A. J., Murphy, A. M., Palukaitis, P. & Carr, J. P. (2002). Virulence and differential local and systemic spread of Cucumber mosaic virus in tobacco are affected by the CMV 2b protein. Mol Plant Microbe Interact 15, 647–653.[Medline]
Szittya, G., Molnár, A., Silhavy, D., Hornyik, C. & Burgyán, J. (2002). Short defective interfering RNAs of tombusviruses are not targeted but trigger post-transcriptional gene silencing against their helper virus. Plant Cell 14, 359–372.
Takeshita, M., Shigemune, N., Kikuhara, K., Furuya, N. & Takanami, Y. (2004). Spatial analysis for exclusive interactions between subgroups I and II of Cucumber mosaic virus in cowpea. Virology 328, 45–51.[CrossRef][Medline]
Thomas, C. L., Jones, L., Baulcombe, D. C. & Maule, A. J. (2001). Size constraints for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vector. Plant J 25, 417–425.[CrossRef][Medline]
Valkonen, J. P. T., Rajamäki, M.-L. & Kekarainen, T. (2002). Mapping of viral genomic regions important in cross-protection between strains of a potyvirus. Mol Plant Microbe Interact 15, 683–692.[Medline]
Van Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L., Carstens, E., Estes, M., Lemon, S., Maniloff, J., Mayo, M. A., McGeoch, D. & other authors (2000). In Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press.
Voinnet, O. (2001). RNA silencing as a plant immune system against viruses. Trends Genet 17, 449–459.[CrossRef][Medline]
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 U S A 96, 14147–14152.
Wang, J.-L., Turgeon, R., Carr, J. P. & Berry, J. O. (1993). Carbon sink-to-source transition is coordinated with establishment of cell-specific gene expression in a C4 plant. Plant Cell 5, 289–296.[Abstract]
Wintermantel, W. M. & Zaitlin, M. (2000). Transgene translatability increases effectiveness of replicase-mediated resistance to cucumber mosaic virus. J Gen Virol 81, 587–595.
Yang, S.-J., Carter, S. A., Cole, A. B., Cheng, N.-H. & Nelson, R. S. (2004). A natural variant of a host RNA-dependent RNA polymerase is associated with increased susceptibility to viruses by Nicotiana benthamiana. Proc Natl Acad Sci U S A 101, 6297–6302.
Yeh, S.-D. & Gonsalves, D. (1984). Evaluation of induced mutants of papaya ringspot virus for control by cross protection. Phytopathology 74, 1086–1091.
Zaitlin, M., Anderson, J. M., Perry, K. L., Zhang, L. & Palukaitis, P. (1994). Specificity of replicase-mediated resistance to cucumber mosaic virus. Virology 201, 200–205.[CrossRef][Medline]
Zhang, X., Yuan, Y.-R., Pei, Y., Shih-Shun Lin, S.-S., Tuschl, T., Patel, D. J. & Chua, N.-H. (2006). Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes Dev 20, 3255–3268.
Received 3 May 2007;
accepted 22 June 2007.
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