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1 Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences (SLU), PO Box 7080, SE-750 07 Uppsala, Sweden
2 Department of Applied Biology, PO Box 27, FIN-00014 University of Helsinki, Finland
Correspondence
Jari P. T. Valkonen
jari.valkonen{at}helsinki.fi
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ABSTRACT |
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Present address: Institute of Pathology, Case Western Reserve University, 2085, Adelbert Road, Cleveland, OH 44106, USA. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Voinnet, 2005). Strands of the resultant small interfering dsRNA molecules (siRNAs; 21–25 nt) are incorporated into a multi-subunit complex, the RNA-induced silencing complex (RISC), which specifically degrades RNA sequences sharing similarity with the inducing dsRNA. The newly produced siRNA may also be used to amplify the RNA-silencing reaction further by the action of host-encoded RNA-dependent RNA polymerase (Dalmay et al., 2000b; Schwach et al., 2005). Consequently, following local induction, RNA silencing spreads systemically (Palauqui et al., 1997; Voinnet & Baulcombe, 1997) via plasmodesmata and phloem, following the long-distance movement route used by viruses (Voinnet et al., 1998; Tournier et al., 2006). The messenger that carries the target specificity is not yet fully resolved. However, many lines of evidence suggest that it may be one of the siRNA species (Hamilton & Baulcombe, 1999; Hamilton et al., 2002; Himber et al., 2003; Shaharuddin et al., 2006).
Successful antiviral defence via RNA silencing may result in recovery of the plant from virus infection. In recovered plants, the new leaves develop free of symptoms and contain little or no detectable amounts of the virus. This is often found in transgenic plants that express viral sequences, e.g. those derived from potyviruses (Lindbo & Dougherty, 1992a, b; Dougherty et al., 1994; Swaney et al., 1995; Guo & Garcia, 1997). Sometimes recovery occurs only in limited areas of the leaf tissue, which results in so-called dark-green islands (DGI). These recovered leaf areas appear healthy and darker green than the surrounding virus-infected tissues (Matthews, 1991; Moore et al., 2001).
The antiviral-defence pathways based on RNA silencing can also be utilized for targeted, systemic suppression of host-gene expression (Fusaro et al., 2006). Kumagai et al. (1995) discovered that infection with a recombinant virus carrying a heterologous sequence corresponding to a host gene resulted in post-transcriptional silencing of the gene. Since then, virus-induced gene silencing (VIGS) (Baulcombe, 1999) has become a popular tool in studies aiming to understand functional roles of plant genes (e.g. Ruiz et al., 1998; Ratcliff et al., 2001; Liu et al., 2002a, b). Replication of the VIGS vector induces primary gene silencing, which targets the homologous region of the host mRNA and triggers host-directed secondary silencing reactions. They involve transitivity, i.e. spreading of silencing to the non-overlapping (non-homologous) part of the host transgene mRNA, as revealed by analysis of the accumulating siRNA (Vaistij et al., 2002; Himber et al., 2003).
It is intriguing that VIGS can suppress host-gene expression efficiently, despite the presence of proteins that viruses produce for suppression of RNA silencing (reviewed by Voinnet, 2005). Helper-component proteinase (HC-Pro) produced by potyviruses (genus Potyvirus) was the first RNA-silencing suppression (RSS) protein discovered (Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschau & Carrington, 1998). This strong RSS protein suppresses silencing by binding siRNA (Lakatos et al., 2006). Potyviruses have not been used for VIGS because it is anticipated that an efficient VIGS vector should be devoid of strong RSS functions, such as those of HC-Pro. Indeed, expression of HC-Pro is used to alleviate silencing of overexpressed genes in plants, to enhance transient expression of recombinant proteins (Johansen & Carrington, 2001; Mallory et al., 2002).
Despite the aforementioned reservations, the aim of this study was to test whether potyviruses could be used as VIGS vectors. Previous studies had shown that plants transformed with sequences of potyviruses (Lindbo & Dougherty 1992a, b; Dougherty et al., 1994; Guo & Garcia, 1997), including those of potato virus A (PVA) (Moore et al., 2001; Savenkov & Valkonen, 2002; Germundsson & Valkonen, 2006), recover from infection with the homologous potyvirus. These studies indicated that silencing by the host can overcome the silencing suppression enforced by the potyvirus. Many studies on VIGS and RNA silencing have utilized Nicotiana benthamiana as the model system, due to the efficient systemic silencing achieved in this plant species (e.g. Voinnet & Baulcombe, 1997; Liu et al., 2002a, b; Tournier et al., 2006). In this study, transgenic N. benthamiana (line 16c), which shows strong constitutive expression of the green fluorescent protein (GFP) (Brigneti et al., 1998) and allows non-disruptive monitoring of the silencing process, was used for experiments. The data showed that PVA can be used for VIGS. A peculiar recovery phenotype in the PVA-infected 16c plants provided further insight into systemic silencing, antiviral defence and formation of DGI.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) that contains the gfp gene inserted close to the N terminus of the P1-encoding region (Fig. 1). The insertion site was originally created by using Mu transposon-mediated mutagenesis (Kekarainen et al., 2002) that introduced a novel NotI restriction site and a few additional, identical nucleotides flanking the NotI site [for details, see the study by Rajamäki et al. (2005)]. In this PVA chimera, GFP is translated as part of the viral polyprotein during infection and subsequently released from the P1 sequence at the flanking, engineered consensus cleavage sites for the viral NIaPro proteinase (Rajamäki et al., 2005). M14-pGFPp was digested by using the unique restriction sites for ApaI and AgeI (Fig. 1). The 3'-proximal genomic fragment thus released was replaced with the corresponding fragment from the highly virulent PVA cDNA pBUIII (Paalme et al., 2004). The resultant chimera (pBUIII-pGFPp) was anticipated to be more virulent than M14-pGFPp based on previous data obtained with different PVA chimeras (Paalme et al., 2004).
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), were grown from seeds for experiments under controlled conditions in growth chambers (Fi-totron 600H; Fisons Environmental Equipment) at a night/day temperature of 19/21 °C, 75 % relative humidity and a 16 h photoperiod. Plants were fertilized weekly with a 1.0 % (N : P : K=8 : 4 : 6) fertilizer.
Virus inoculation.
Plants were inoculated on the first fully expanded leaf when they were 5 weeks old. Inoculation with the viral cDNA was done by using particle bombardment as described previously (Rajamäki & Valkonen, 1999). Mechanical inoculation was done by using sap extracted from leaves infected systemically by pBUIII-pGFPp. The leaves were ground in distilled water and the sap was rubbed onto the first true, fully expanded leaf dusted with carborundum. Five independent experiments were carried out under the same experimental conditions. For the first 21 days post-inoculation (p.i.), the symptoms and GFP fluorescence in the plants were documented daily and, for the following 4 weeks, three times per week. The leaves were numbered and documented by a digital camera (Camedia E-10, 4.0 megapixels; Olympus) under normal light and illuminated with a UV lamp (Blak-Ray non-UV semiconductor inspection lamp, model B 100 AP; Ultra-Violet Products Ltd) at each time of observation.
RNA extraction and Northern and dot-blot hybridizations.
Total RNA was extracted from leaves (100 mg) with an RNeasy Mini kit (Qiagen). Extraction of high-molecular-mass RNA (mRNA) and low-molecular-mass RNA (siRNA) from leaf tissue (400 mg), preparation of the [
-32P]UTP-labelled probes for detection of the gfp gene, the positive strand of the 5' non-translated region (5'-NTR) and the CP-encoding region of PVA, and Northern blot analysis of GFP mRNA and GFP- and PVA-derived siRNA were done as described in detail previously (Kreuze et al., 2005). For detection of the negative strand (replicative form) of PVA RNA in infected tissues, 1 µg total RNA was dotted onto a nylon membrane (Hybond-N; Amersham Biosciences) and stained with methylene blue to check the loading of equal amounts of RNA. The plasmid pPCRII (Invitrogen) containing the CP-encoding region of PVA (Spetz & Valkonen, 2004) was restricted with ApaI and the probe complementary to the negative RNA strand of the PVA CP-encoding region was synthesized by SP6 RNA polymerase (Promega). The probe was labelled with digoxigenin (DIG) 11-UTP by using a DIG Northern starter kit (Roche). Hybridization, washing and detection of signals using anti-DIG alkaline phosphatase-conjugated Fab fragments were done according to the manufacturer's instructions (Roche). The membrane was exposed in a luminescent image analyser (LAS 3000; Fujifilm).
RT-PCR.
Viral RNA was reverse-transcribed by using RevertAid Moloney murine leukemia virus reverse transcriptase (Fermentas) and random hexamer primers [(dN6)] according to the manufacturer's instructions. The cDNA was synthesized from total RNA and amplified by PCR using the PVA-specific forward primer 61475, designed to the 5' end of the viral RNA (nt 3–23), and the reverse primer FO19R, hybridizing to the P1-encoding region (nt 380–400) (Rajamäki et al., 2005). The amplified products were sequenced directly or, alternatively, analysed by agarose-gel electrophoresis, isolated from the gel, cloned and sequenced.
Serological detection of PVA.
Leaf tissue was sampled, weighed and ground at 1 g in 3 ml ELISA extraction buffer. PVA was detected by double-antibody sandwich (DAS)-ELISA with a monoclonal antibody (mAb) and an alkaline phosphatase-conjugated mAb to PVA (mAb 58/0; Adgen), using p-nitrophenyl phosphate as the substrate, as described previously (Rajamäki et al., 1998). Known amounts of purified PVA virions were included for comparison, to estimate viral concentrations in the samples.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) (Fig. 1
). PVA–GFP is similar to pBUIII, a chimera in which the 3'-proximal part of the genome of isolate B11 is replaced with that of isolate U (Paalme et al., 2004). Replacement of the 3' part of the genome was expected to increase virulence, as shown with pBUIII (Paalme et al., 2004), and to compensate for diminished virulence caused by the GFP insertion in P1 (Rajamäki et al., 2005). PVA–GFP expressed functional GFP, because GFP is separated from the viral polyprotein by activity of the PVA NIaPro (Rajamäki et al., 2005). Indeed, inoculation of the virus chimera to 5-week-old wt N. benthamiana plants resulted in systemic infection by 7 days p.i., as revealed by green fluorescence observed under UV light. The systemically infected leaves displayed mild mosaic symptoms and slight malformation and contained high titres of the PVA CP antigen [20 µg (g leaf tissue)–1] as detected by ELISA.
Following inoculation of the lowest fully developed leaf in 5-week-old plants, systemic infection was monitored and documented by using a digital camera until 70 days p.i. The leaves that existed above the inoculated leaf at the time of inoculation and the new leaves were numbered. The infection process in all of them was studied carefully. The systemic-infection process was divided into phenotypically distinguishable stages (Fig. 2) at which leaves were sampled for molecular analysis. Five similar, independent experiments were carried out, each containing ten 16c and ten non-transgenic (wt) plants. The data are summarized in Table 1.
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), the 16c plants were uniformly green under UV light, owing to the transgene-expressed GFP. Inoculation with PVA–GFP did not cause any detectable phenotypic changes in the green fluorescence of the inoculated leaf in line 16c. The inoculated leaves of the wt plants remained red under UV light, but green fluorescence appeared in the petiole of the inoculated leaf by 7 days p.i. (Fig. 3).
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) indicated expression of GFP transgene mRNA and only little GFP-specific siRNA (Fig. 4a), but no viral RNA or siRNA (Fig. 4b, c). In contrast, the inoculated leaf (sample 1) accumulated considerable amounts of GFP siRNA (Fig. 4a) and contained detectable amounts of viral RNA (Fig. 4b, c), as well as siRNA derived from the length of the viral genome (Fig. 4b).
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). Leaves 3–5 displayed systemic infection at 7 days p.i. (stage II in Fig. 2), but the phenotypes of leaves 3 and 4 differed from that of leaf 5 in 16c plants. In leaves 3 and 4, the basal part of the leaf became brighter green than the tip (Fig. 2; Table 1), due to additional expression of GFP from the PVA vector. This was not seen as additional accumulation of GFP transgene mRNA, as detected by Northern blot analysis (sample 3 versus 4, Fig. 4a), because the gfp in PVA–GFP is part of the chimeric viral genome (approx. 10 kb). Accumulation of the viral RNA was detected in the basal part by dot-blot and RT-PCR (sample 3, Fig. 4b, c), but not in the tip that remained dull green (sample 4, Fig. 4b, c). The initial difference in brightness of fluorescence between the basal part and the tip faded gradually. In wt plants, GFP fluorescence appeared only in the basal part of the leaf (Fig. 3).
In contrast to leaves 3 and 4, the basal part of leaf 5 in 16c plants expressed initial signs of GFP silencing at 7 days p.i. Silencing was detected visually by disappearance of the green fluorescence from veins and the tissues adjacent to them (Table 1). Consequently, the tissues appeared purple or red under UV light, owing to the autofluorescence of chlorophyll. Molecular analysis of leaf 5 in 16c plants revealed a high accumulation of GFP transgene mRNA and siRNA (sample 5, Fig. 4a) and also accumulation of viral RNA and siRNA (Fig. 4b, c). In wt plants, the basal part of leaf 5 showed strong GFP fluorescence from the PVA–GFP vector (Fig. 3).
At 21 days p.i. (stage III in Fig. 2), leaf 5 in 16c plants still showed signs of GFP silencing only in the basal part, whereas the veins and adjacent tissues in the whole leaf 6 were affected by GFP silencing (Table 1; Figs 2, 3). In addition, leaf 6 in 16c plants displayed green vein banding under visible light, the pattern of which matched that in tissues appearing red under UV light (i.e. tissues in which the gfp transgene was silenced) (Fig. 3). Leaf 6 of 16c plants contained only barely detectable amounts of GFP transgene mRNA, but an abundance of the corresponding siRNA (sample 6, Fig. 4a). Viral replicative (negative-strand) and genomic RNA, as well as virus-derived siRNA, were detected readily (Fig. 4b, c). No mutants of PVA–GFP that would have lost the GFP insert were observed (Fig. 4c). In the wt plants, the whole leaf blade in leaf 6 expressed GFP fluorescence generated by PVA–GFP (Fig. 3).
Phenotypically uniform systemic silencing
GFP expression in leaves 7–10 was silenced strongly in 16c plants and the leaves appeared uniformly red under UV light at 28 days p.i. (Table 1; Fig. 2). GFP-specific siRNA accumulated in very high amounts, but low to modest amounts of GFP transgene mRNA were also detectable (sample 7, Fig. 4a). It was unexpected to find that the amounts of GFP mRNA were higher in leaf 9 than in leaf 6 (Fig. 4a), but these results were reproduced in each experiment. Perhaps in leaf 6, the earlier-synthesized GFP protein retains integrity and exhibits fluorescence for some time after induction of silencing, whereas the newly synthesized GFP mRNA is degraded. In leaf 9, the mRNA levels may not reach the levels needed for accumulation of visible amounts of the GFP protein because silencing affects the leaf from its early stages of development.
PVA genomic RNA (Fig. 4c) and PVA-specific siRNA (Fig. 4b) were also detected in leaves 7–10, but the virus no longer contained the entire GFP insert. The RT-PCR amplification products were shorter (lane 7 in Fig. 4c) than expected for a virus carrying the full-length insert (lane ‘pi’ in Fig. 4c) and similar or slightly larger than those obtained from the pBUIII-pGFPp plasmid from which the gfp insert had been removed (lane ‘pe’ in Fig. 4c). During the next 7–10 days (up to 38 days p.i.), all new leaves (nos 7–15) of 16c plants appeared red under UV light (stage IV in Fig. 2; Table 1).
Appearance of viral deletion mutants and partial loss of silencing
Green fluorescent patches of tissue begun to emerge in the newest leaves (nos 13–15) by 35 days p.i. (stage V in Fig. 2) (Table 1; Figs 2, 3). These leaves contained modest amounts of GFP transgene mRNA and high amounts of GFP siRNA (sample 8, Fig. 4a). Amounts of the replicative PVA RNA were higher and the amounts of PVA-derived siRNA much higher than in leaves sampled at earlier time points (Fig. 4b). The virus carried only fragments of the gfp insert (Fig. 4c). The PCR products obtained from 15 leaves at positions 9–14 were cloned and sequenced. Products that appeared slightly larger (lane 8 in Fig. 4c) than the insert-less control (lane ‘pe’, Fig. 4c) in agarose-gel electrophoresis contained parts of the gfp insert. The shortest amplification products had lost the gfp insert and up to 45 nt of the P1-encoding region (data not shown). Despite the deletions, all sequences retained the original reading frame of the PVA polyprotein.
GFP silencing pattern corresponding to DGI
One week later (42 days p.i., stage VI), the newest leaves (nos 16–18) displayed an irregular red–green pattern under UV light (Table 1; Fig. 2). When these leaves were observed under visible light, they were found to contain DGI that matched spatially with the red areas observed under UV light (Fig. 3). These leaves also showed stronger symptoms of PVA infection than before, similar to the top leaves in the wt plants. This was expected because the leaves in 16c and wt plants, at this time, were infected exclusively with the deletion mutants. The gfp inserted in the P1 region of PVA–GFP diminishes accumulation of HC-Pro, reduces virulence and alleviates symptoms, whereas loss of the insert restores the high virulence and severe symptoms (Rajamäki et al., 2005). The green and red tissues were separated with a scalpel under UV light as carefully as possible and subjected to molecular analysis. In the green tissue, the amounts of GFP transgene mRNA were modest (sample 9, Fig. 4a), whereas in red tissues, GFP mRNA was barely detectable (sample 10, Fig. 4a). In the red tissue, accumulation of GFP siRNA was somewhat higher than in the green tissues (Fig. 4a) (more contrasting differences in amounts of the various RNAs could probably be obtained if it were possible to separate the red and green tissues more accurately). Accumulation of viral replicative RNA was very high in green tissues, but low in the red tissues (Fig. 4b). Similarly, virus-derived siRNA accumulated to very high amounts in green tissues, but only to low amounts in red tissues (Fig. 4b).
PVA–GFP deletion mutants lacking the gfp insert became increasingly abundant in leaves 13–15 of the wt plants during the course of systemic infection, which occurred at the same time in the 16c plants. Consequently, the leaves at position 16 and above no longer showed GFP fluorescence. However, they showed strong PVA symptoms and contained DGI (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). There are several previous reports on transgenic plants that express potyviral sequences and recover from infection with the homologous virus by the RNA silencing-based antiviral-defence mechanism, despite the RSS protein HC-Pro encoded by these viruses (e.g. Lindbo et al., 1993; Simón-Mateo et al., 2003; Van Den Boogaart et al., 2004; Germundsson & Valkonen, 2006). In an extreme example, the transgenic plants expressed high titres of HC-Pro, but still recovered from infection with the homologous virus (PVA) (Savenkov & Valkonen, 2002). These studies show that potyviruses cannot prevent silencing-based antiviral defence in all situations, despite the strong RSS protein that they produce. Hence, they can be used for VIGS.
Many studies have employed strains of the species Tobacco mosaic virus (TMV, genus Tobamovirus) (Kumagai et al., 1995), Potato virus X (PVX, genus Potexvirus) (Ruiz et al., 1998) or Tobacco rattle virus (genus Tobravirus) (Ratcliff et al., 2001) as VIGS vectors. These vectors express the heterologous sequences not only as part of the new copies of the viral genomic RNA, but also in subgenomic RNA that is synthesized during viral replication. Hence, potentially very high amounts of the silencing-inducing RNA are produced. In this study, silencing was induced exclusively by the replicating viral genomic RNA, as potyviruses do not encode subgenomic RNAs.
PVA–GFP caused mild symptoms, which are due to the gfp insert in P1 (Rajamäki et al., 2005). Similarly, an insert in the P1 sequence of a P1/HC-Pro transgene abolishes the developmental defects that are observed in plants transformed with the wt P1/HC-Pro sequence of Tobacco etch virus (Mallory et al., 2002). The alleviated symptoms may be associated with reduced RSS efficiency, both of which are advantageous changes in the properties of a virus to be used for VIGS. The PVX amplicon used for VIGS lacks a part of the RSS protein p25 (Mallory et al., 2002). Reduced virulence and alleviated symptoms are often observed with potyviruses that carry foreign inserts in the genome (e.g. Guo et al., 1998; German-Retana et al., 2000). Therefore, it seems that insertion of foreign sequences into potyviruses alters the viral properties for the benefit of VIGS.
The leaf inoculated with PVA–GFP contained rather high amounts of gfp-specific siRNA, which showed that the replicating PVA–GFP induced silencing of the GFP transgene mRNA owing to sequence homology. The GFP-specific siRNA could be derived from the vector virus and GFP transgene mRNA in the inoculated leaf because the PVA–GFP genome also became targeted by silencing. This was indicated by siRNAs that were specific to the 5'- and 3'-proximal parts of the genomic (+) strand of PVA RNA and that accumulated in equal amounts, which revealed that silencing affected the length of the whole viral genome, possibly due to transitivity (Vaistij et al., 2002; Himber et al., 2003). wt RNA viruses, including those of the species Cymbidium ringspot virus (CymRSV, genus Tombusvirus), PVX and TMV, are targeted by silencing in non-transgenic plants (Hamilton & Baulcombe, 1999; Molnár et al., 2005). The siRNAs of CymRSV accumulate from the length of the whole viral genome and are mostly derived from highly structured regions of the (+) single-stranded RNA (Molnár et al., 2005). It is therefore possible that different parts of the PVA–GFP genome also became targeted by silencing by this latter mechanism.
The first signs of systemic silencing of the GFP transgene were observed in the fifth leaf above the inoculated leaf. Systemic spread of silencing (Yoo et al., 2004; Tournier et al., 2006) and virus infection (Roberts et al., 1997; Santa Cruz, 1999) follow the source–sink transition in which import ceases and export is initiated at the transition boundary (Turgeon, 1989). Systemic spread and spatial distribution of silencing in 16c plants and movement of PVA–GFP in wt plants were consistent with the aforementioned model. All younger leaves above leaf 5 were also affected by GFP transgene silencing, leaves 7–12 having fully lost visible GFP fluorescence. Silencing was not complete, as signals for GFP mRNA were detected in these leaves, but it was efficient enough for a drastic phenotypic alteration in GFP expression. Furthermore, the silenced leaves were free of viral symptoms. Virus-derived siRNA and low amounts of replicating PVA were detected, indicating that the leaves expressed silencing-based resistance to PVA. Until this point, the phases of the silencing process that targeted the GFP transgene and the VIGS vector closely resembled those described for PVX–GFP in N. benthamiana (Ruiz et al., 1998).
However, when mutants of PVA–GFP with deletion of the gfp insert were generated during viral replication, their accumulation released GFP expression in leaves at position 9 and above. This was observed as an appearance of green fluorescent spots and small areas in the leaves that were previously silenced. This new phase, including insert-less mutants and reversal of silencing, did not occur in the GFP-transgenic plants infected with PVX–GFP (Ruiz et al., 1998). Appearance of deletion mutants of PVA–GFP was observed at the same time in the 16c and wt plants. Thus, the loss of insert was not associated with GFP silencing, in contrast to a previous study in which chimeric PVX lost the heterologous sequence faster in the transgenic plants that expressed the same sequence (Barajas et al., 2006). Probably, when the amount of silencing-inducing PVA–GFP RNA decreased gradually due to the increased proportion of deletion mutants in the viral population, the RSS functions of HC-Pro could overcome the maintenance of silencing locally. This process took a new turn in the top leaves of 16c plants infected exclusively with the viral deletion mutants. These leaves expressed a peculiar, mosaic-like red–green pattern under UV light. The pattern was observed as soon as the tip leaves opened. When leaves expanded, it was apparent that the red tissue corresponded to DGI observed under visible light. Development of DGI also took place in the top leaves of wt plants infected with the deletion mutants and was not associated with GFP transgene silencing. We found, by photographing the same leaves daily over several weeks, that DGI were firmly established.
DGI are localized areas of virtually virus-free leaf tissue that develop only in leaves whose cells are actively dividing. They are darker green than the surrounding chlorotic, heavily virus-infected tissues and are resistant to superinfection with the same virus (Reid & Matthews, 1966; Matthews, 1991). DGI form based on virus-induced RNA silencing (Moore et al., 2001). It is apparent that silencing of the GFP transgene in DGI was induced by a systemic signal transported from lower leaves because the viruses in these leaves were exclusively deletion mutants devoid of the GFP insert. Systemic spread of silencing in 16c plants showed that HC-Pro was unable to interfere with it, as reported in previous studies (Mallory et al., 2001, 2003). Silencing of gfp was maintained in DGI because accumulation of PVA was inhibited in them by a virus-resistance mechanism. In tissues surrounding DGI, strong GFP expression from the transgene was possible, owing to RSS caused by replicating PVA.
However, it is not known whether the DGI were induced locally as a response to PVA replication or by systemic movement of the silencing signal from lower leaves (Moore et al., 2001). The latter hypothesis seems plausible. The systemic signal for silencing may be one of the siRNA species (Hamilton & Baulcombe, 1999; Hamilton et al., 2002; Himber et al., 2003; Yoo et al., 2004; Dunoyer et al., 2006; Shaharuddin et al., 2006) and its transport occurs via the vascular system (Tournier et al., 2006). Because HC-Pro enforces RSS by binding siRNA, it cannot inhibit the activity of assembled RISC (Lakatos et al., 2006). Incorporation of the transported PVA-specific siRNA to RISC in the developing tip leaves prior to virus entry, or transport of pre-assembled RISC from lower leaves, would allow initiation of silencing from the beginning of virus infection and despite HC-Pro. In the localized areas where this process is initiated, silencing is expected to accelerate fast and to surpass the RSS driven by the virus. Consequently, expansion of the leaf makes DGI visible. This model is supported by observation of the red–green mosaic pattern as soon as the tip leaves opened and frequent localization of the DGI along the veins. It is not understood how the virus-resistant state in DGI is created and maintained in the absence of a virus-homologous host gene. In transgene silencing associated with recovery from virus infection, methylation of the transgene often leads to the maintenance stage of silencing (Dalmay et al., 2000a). Future studies should compare gene expression in DGI with that in the surrounding tissues, to provide further insights on spatial induction of the silencing-based antiviral defence.
Taking these results together, this study allowed us to monitor the dynamic, systemic, potyvirus-induced silencing process in a non-disruptive manner. The silencing-inducing GFP sequence was not pivotal to viral infectivity, in contrast to previous studies that have described recovery from potyvirus infection in plants that were transformed with sequences of the homologous virus. Therefore, appearance of the insert-less, virulent mutants added a new dimension to the dynamic interplay between silencing and its suppression.
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ACKNOWLEDGEMENTS |
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Received 15 February 2007;
accepted 24 April 2007.
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