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Viroids, small non-coding pathogenic RNAs, are able to induce RNA silencing, a phenomenon that has been associated with the pathogenesis and evolution of these small RNAs. It has been recently suggested that viroids may resist this plant defense mechanism. However, the simultaneous degradation of non-replicating full-length viroid RNA, and the resistance of mature forms of viroids to RNA silencing, have not been experimentally demonstrated. Transgenic Nicotiana benthamiana plants expressing a dimeric form of Hop stunt viroid (HSVd) that have the capability to cleave and circularize this viroid RNA were used to address this question. A reporter construct, consisting of a full-length HSVd RNA fused to GFP-mRNA, was agroinfiltrated in these plants and its expression was suppressed. Interestingly, both circular and linear HSVd molecules were stable and able to traffic through grafts in these restrictive conditions, indicating that the mature forms of HSVd are able, in some way, to resist the RNA-silencing mechanism. The observation that a full-length HSVd RNA fused to GFP-mRNA, but not circular and/or linear viroid forms, was fully susceptible to RNA degradation strongly suggests that structures adopted by the free mature monomer protect the pathogenesis-associated forms of the viroid from RNA silencing.
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RNA silencing is an evolutionarily conserved surveillance system that regulates gene expression in eukaryotes. This term refers to a set of related mechanisms found in animals (RNA interference, RNAi), fungi (quelling) and plants (post-transcriptional gene silencing, PTGS) (Baulcombe, 2002; Brodersen and Voinnet, 2006; Kim and Nam, 2006; Nakayashiki, 2005). This phenomenon, based on the sequence-specific degradation of single-stranded RNA (ssRNA), is directed by double-stranded RNA (dsRNA) or self-complementary hairpin RNA (hpRNA; Baulcombe, 2002; Zamore, 2001). These RNAs are cleaved by an RNase III-like enzyme known as Dicer to generate small (21–25 nt) RNAs, termed small interfering RNAs (siRNAs) (Bernstein et al., 2001; Hamilton and Baulcombe, 1999; Hamilton et al., 2002). siRNAs guide a nuclease complex, referred to as the RNA-induced silencing complex (RISC), to target RNAs for degradation (Hammond et al., 2000; Zamore, 2001). Based on these findings, the presence of siRNA is accepted as a hallmark of RNA silencing.
Post-transcriptional gene silencing plays an important role as an ancient cellular defense system in plants, acting against diverse molecular parasites including transposons, transgenes and pathogenic RNAs (Voinnet, 2001; Zamore, 2001). To counteract the antiviral effect of PTGS, many viruses have developed counterdefensive strategies by encoding proteins that block one or more steps of the silencing pathway (Llave et al., 2000; Voinnet, 2001).
In recent years, the presence of viroid homologous siRNAs in infected plants has been taken as indirect evidence that viroids induce PTGS (Itaya et al., 2001; Markarian et al., 2004; Martínez de Alba et al., 2002; Papaefthimiou et al., 2001). Stronger evidence obtained by transgenic models support the idea that viroids and/or their replication intermediates induce a phenomenon, termed viroid-induced RNA silencing (VdIRS), capable of degrading Potato spindle tuber viroid (PSTVd) fragments fused to an mRNA reporter (Vogt et al., 2004). In addition, in a recent work, it has been shown that structured PSTVd-RNAs, transiently expressed in Nicotiana benthamiana protoplasts, are resistant to RISC-mediated degradation because of their compact structure (Itaya et al., 2007). Thus, because of their structural compactness, viroid molecules might have evolved to escape this host defense pathway, as suggested by Wang et al. (2004).
These findings provide a unified picture in which the non-coding viroid RNA is at the same time inductor, target and evader of the RNA-silencing phenomenon (Flores et al., 2005; Itaya et al., 2007; Vogt et al., 2004; Wang et al., 2004). However, the simultaneous degradation of non-replicating full-length viroid RNA sequences, together with the generation and resistance of biological forms of the viroid to RNA silencing, has never strictly been experimentally validated.
Here, we demonstrate that in N. benthamiana transgenic plants, the biological forms of Hop stunt viroid (HSVd) can resist the RNA silencing-mediated degradation, whereas identical full-length HSVd sequences fused to the mRNA GFP could not. In addition, these biological forms of HSVd were able to traffic to distal parts of the plant through the silenced environment. Our results also suggest that the highly compact secondary structure of the free mature monomeric HSVd molecules offers resistance or increased tolerance to this plant defense mechanism.
Expression of chimeric HSVd-GFP into N. benthamiana plants
To demonstrate that HSVd-RNA expression induces RNA silencing, a HSVd-GFP reporter was constructed by fusing a full-length HSVd cDNA, in which an initiation codon was inserted (ATG-HSVd), with a properly digested GFP cDNA. The fused HSVd-GFP fragment was then inserted in the pMOG 800 binary vector (Knoester et al., 1998; Figure 1a). The correct transcriptional fusion of the HSVd-GFP fragments was checked by DNA sequencing and by in silico translation of the chimeric mRNA (Figure S1). The functionality of this reporter was studied by comparing its transient expression with that of the unmodified GFP mRNA in N. benthamiana plants.
The fluorescence signal of the free GFP and the HSVd-GFP was clearly observed at 3 days post infiltration (dpi), as seen in Figure 1b (panels 1 and 2), demonstrating that the HSVd-GFP reporter is functional. The emission range of both GFP and HSVd-GFP constructions were similar (data not shown), indicating that the fluorescence of the GFP is not substantially modified by the HSVd fusion.
To analyze the GFP and HSVd-GFP expression, total proteins were extracted from agroinfiltrated and mock-agroinfiltrated leaves, and were analyzed by western blot assays. As observed in Figure 1c (panel II), the unmodified GFP (lane 1) and HSVd-GFP (lane 2) showed a comparable signal, demonstrating that the expression level of the HSVd-GFP reporter is similar to that of the unmodified GFP. In addition, although their relative electrophoretic mobility in 10% SDS-PAGE was similar (Figure 1c, panel II), a more detailed analysis of the chimeric HSVd-GFP both at the mRNA and protein level confirmed the expected differences with respect to the unmodified GFP (Figure S2).
HSVd RNA-induced RNA silencing
HSVd-transgenic N. benthamiana plants carrying a dimeric HSVd cDNA able to be transcribed and processed into the biologically active monomeric circular and linear forms (Gómez and Pallás, 2006), were used to analyze the HSVd RNA-induced RNA silencing. Plants (20-days-old) of two independent lines (HSVd2x-2 and HSVd2x-4) of HSVd-transgenic N. benthamiana were agroinfiltrated with the HSVd-GFP reporter and analyzed by confocal microscopy at 3 dpi. Expression of the HSVd-GFP reporter was suppressed in the HSVd-transgenic plants in contrast with the same reporter infiltrated into untransformed N. benthamiana plants, which showed a normal fluorescence level (Figure 2a, compare panel 3 with panels 6 and 9). The fluorescence of the agroinfiltrated unmodified GFP, used as a positive control, was similar in both transgenic and untransformed plants (Figure 2a, panels 2, 5 and 8).
To gain additional evidence of the lack of HSVd-GFP reporter expression, total proteins extracted from agroinfiltrated leaves were analyzed by western blot assays. The HSVd-GFP protein was not detected in the HSVd-transgenic N. benthamiana leaves agroinfiltrated with the HSVd-GFP reporter (Figure 2b, panel I, lanes 6 and 9), whereas it was detected in untransformed N. benthamiana leaves (Figure 2b, panel I, lane 3). The unmodified GFP control was normally detected in HSVd-transgenic and untransformed N. benthamiana leaves (Figure 2b panel I, lanes 2, 5 and 8). Identical results were obtained when 120-day-old HSVd-transgenic N. benthamiana leaves agroinfiltrated with the HSVd-GFP reporter were analyzed (Figure S3), indicating that the phenomenon that blocks the HSVd-GFP reporter expression in HSVd-transgenic N. benthamiana plants is long-lasting.
The levels of mRNA in the agroinfiltrated leaves, where the suppression of HSVd-GFP expression had been previously confirmed (Figure 3a, panels 4 and 6), were analyzed. Northern blot assays showed that the chimeric HSVd-GFP mRNA was absent in the HSVd-transgenic plants (Figure 3b; panel II, lanes 4 and 6), but not in the mock-transformed plants agroinfiltrated with the HSVd-GFP reporter (Figure 3b; panel II, lane 2). Similar GFP mRNA levels were observed in HSVd-transgenic and mock-transformed plants infiltrated with the unmodified GFP control (Figure 3b; panel II, lanes 1, 3 and 5).
The presence of HSVd-specific siRNAs in the HSVd-transgenic N. benthamiana plants was studied by northern blot assays. These analyses revealed the presence of abundant 20–25-nt-long RNAs, a hallmark of the RNA-silencing process in the HSVd-transgenic plants tested (Figure 3c; panel II, lanes 3 and 4), but not in the untransformed and mock-transformed controls (Figure 3c; panel II, lanes 1 and 2, respectively). By contrast, northern blot hybridization under similar conditions, but with the GFP probe, revealed that no GFP-specific siRNAs were detected (data not shown). These results indicate that the lack of expression of the HSVd-GFP reporter in the HSVd-transgenic N. benthamiana plants is caused by the RNA degradation of its mRNA, by means of HSVd RNA-induced RNA silencing.
The biological forms of the viroid can resist the HSVd RNA-induced RNA silencing
Once established that the HSVd RNA expression induces the RNA-silencing phenomenon in HSVd-transgenic N. benthamiana plants, and that this mechanism triggers the degradation of a non-replicating full-length HSVd RNA fused to the GFP mRNA, actively transcribed, we attempted to determine if the mature forms of HSVd, associated with a viroid infection, were able to resist this plant defense strategy. To address this issue, we compared the viroid levels in HSVd-transgenic N. benthamiana plants previous to agroinfiltration with the viroid levels present after 3 dpi (when the RNA degradation of HSVd-GFP reporter is complete) by northern blot assays. No significant differences in the levels of the mature biological forms of the viroid were observed (Figure 4b; panel II, compare lanes 3 and 5 with lanes 4 and 6), even when the HSVd-GFP reporter, determined by fluorescence observation, was completely silenced (Figure 4a, panels 4 and 6) and the HSVd-specific siRNAs were clearly detected (Figure 4b, panel III, lanes 3–6). These findings indicate that monomeric circular and linear HSVd forms are stable in these extremely restrictive conditions, and strongly suggest that mature forms of HSVd are resistant or less sensitive to RNA silencing, thereby counteracting this plant defence mechanism.
To provide biological evidence that the mature forms of the viroid avoid RNA silencing, we determined if circular and linear monomeric HSVd could move and traffic in silenced plants. To this end, wild-type N. benthamiana scions were grafted onto silenced transgenic N. benthamiana stocks (Figure 5a) and total RNA was analyzed 30 days after grafting. Northern blot assays confirmed previous observations (Gómez and Pallás, 2006) that HSVd is translocated through homogeneric grafts in transgenic N. benthamiana (Figure 5b; panel II, lanes 1 and 2), indicating that mature forms of HSVd can pass across the silenced cytoplasm and enter into the vascular tissue for long-distance transport.
These findings support an emergent view of viroid–host interactions, by which these pathogenic RNAs are capable of inducing the RNA-silencing process while developing a strategy to counteract this plant defense mechanism (Wang et al., 2004). Because viroids lack mRNA activity, both antagonistic mechanisms will be regulated by their characteristic RNA sequence and/or structure (Chang et al., 2003; Wang et al., 2004). Indeed, the possibility that viroids can themselves be inducers, targets and evaders of RNA silencing is an attractive idea. In this scenario, it is important and necessary to obtain a large body of evidence to unambiguously demonstrate the coexistence in plants of these different functions/situations.
Here, we show that: (i) the expression of HSVd-RNA in transgenic plants induces the degradation through RNA silencing of a full-length HSVd RNA fused to GFP mRNA, and (ii) the mature, highly structured forms of the viroid associated with the pathogenic process can overcome this plant defense mechanism.
To demonstrate the ability of HSVd RNA expression to induce the RNA silencing, we designed a transcriptional fused 5′ HSVd-GFP silencing reporter, which was used to agroinfiltrate N. benthamiana transgenic plants expressing and processing dimeric plus-strand HSVd-RNA (Gómez and Pallás, 2006). This test clearly shows that the HSVd-GFP expression, estimated by fluorescence emission or western blot assays, was suppressed in these transgenic plants but not in untransformed controls. The analysis by northern blot of the mRNA levels in agroinfiltrated plants confirmed that the suppression of the HSVd-GFP reporter expression was associated with the absence of its mRNA, which together with the detection of HSVd-specific siRNAs, a hallmark of RNA silencing, in the N. benthamiana HSVd-transgenic plants revealed the existence of HSVd RNA-induced RNA silencing. These findings indicate that in N. benthamiana transgenic plants the HSVd RNA expression induces the RNA-silencing-mediated RNA degradation of a non-replicating full-length HSVd RNA transiently expressed. Although very unlikely, the possibility that siRNAs derived from the nopaline synthase terminator (tNos), used for both the viroid delivery construct and for the HSVd-GFP sensor construct, would trigger transitivity on the HSVd delivery mRNA can not be excluded. Our results are in accordance with a previous report showing that in tobacco transgenic plants, expressing a reporter gene 3′ fused to partial-length PSTVd sequences, the subsequent PSTVd infection activates the degradation of the partial-length recombinant transcript via the siRNAs that result from PSTVd infection (Vogt et al., 2004).
Interestingly, in these restrictive conditions the circular and linear forms of HSVd levels were maintained as stable, indicating that the biological forms of HSVd have developed strategies to counteract the RNA-silencing phenomenon. This is consistent with the hypothesis proposed by Wang et al. (2004) suggesting that viroids would be, in some way, resistant to RNA silencing. However, Wang et al. (2004) observed this resistance with non-replicating PSTVd sequences fused to mRNA GUS (β-glucuronidase), and suggested that the resistance to RNA silencing was intrinsic to the PSTVd sequence. Differences in the secondary structure adopted by the viroid sequence in both (HSVd-GFP and PSTVd-GUS) reporters may explain this discrepancy.
Previous studies suggest a direct role of secondary RNA structure in conferring resistance to RNA silencing (Metzlaff et al., 1997; Szittya et al., 2002). Our results strongly suggest that the secondary structure adopted by free monomeric mature viroid forms confers significant resistance against the RNA degradation mediated by RNA silencing. This idea is in accordance with the results recently reported by Itaya et al. (2007), in which structured PSTVd-RNAs transiently expressed in N. benthamiana protoplasts were resistant to RISC-mediated cleavage. These results are also consistent with previous observations showing that unit-length RNA of PSTVd and ASBVd were resistant in vitro to human Dicer degradation (Chang et al., 2003), and to those that showed that monomeric transcripts of PLMVd are less susceptible to Dicer activity from wheat germ extracts than a partial sequence of 83 nt in size harboring a hairpin structure (Landry and Perreault, 2005). It is worthy to note here that the offspring of genetic crosses between the transgenic viroid-expressing Arabidopsis plants (Daròs and Flores, 2004) with different Dicer-like single mutants (dcl1-7, dcl2-1, dcl3-1 and dcl4-2) did not show any alteration in the levels of viroid RNAs (J. Marqués and J.A. Daròs, personal communication). However, double or even triple Dicer mutant combinations are required to conclusively demonstrate any correlation between viroid accumulation and dependence of Dicer-like activities.
Perhaps the strongest evidence supporting the idea that the mature forms of HSVd resist or escape RNA silencing rely on the observation that the viroid is able to move throughout grafts in silenced HSVd-transgenic N. benthamiana plants, in which monomeric HSVd must leave the nucleus, a potentially ‘safe’ zone (Denti et al., 2004) and pass through the cytoplasm (where RNA silencing is active), for cell-to-cell and long-distance movement. This viroid movement through a highly restrictive zone could be explained considering that mature monomeric HSVd (resistant or less sensitive, to RNA silencing) has the ability to evade this RNA degradation-mediated plant defense mechanism. In addition, as long-distance movement of viroids occurs within the phloem (Gómez and Pallás, 2001;Zhu et al., 2001), a tissue formed by enucleate cells, it is difficult to accommodate the idea of compartmentalization in the nucleus as a general mechanism to evade RNA silencing. In addition, Itaya et al. (2007) recently demonstrated that PSTVd replication was resistant to RNA silencing in silenced tomato plants, and they proposed that this characteristic was largely attributed to the resistance of its secondary structure to RISC-mediated cleavage, rather than to an RNA-based silencing suppressor activity or to subcellular localization. However, the possibility that viroid RNA use the interactions with host proteins (e.g. Gómez and Pallás, 2004) as an alternative strategy to avoid silencing during their movement cannot be ruled out.
In summary, our results demonstrate that in transgenic plants, HSVd RNA can be simultaneously a target and an evader of the RNA silencing, and strongly suggest that, at least during trafficking processes, this resistance is mediated by the highly conserved secondary structure of the mature forms of viroid. The involvement of these viroid RNA characteristics in more complex mechanisms such as pathogenicity effectors and/or evolutionary survival strategies (for and extensive discussion see Daròs et al., 2006; Flores and Pallás, 2006; Flores et al., 2005; Wang et al., 2004) remains an intriguing possibility to be elucidated.
The modified atg-HSVd cDNA fragment was amplified by PCR from a plasmid containing the cDNA of HSVd isolate PR1A (Y09347; Kofalvi et al., 1997) using the Vp/atg-HSVd (5′-ctctctccatgggc ccctctgg-3′) and the Vp/end-HSVd (5′-gctagcgagaggatccgcggc-3′) primers, as previously described (Amari et al., 2001). The amplified atg-HSVd fragment was ligated to GFP cDNA and cloned in a binary plasmid pMog800 under the control of the Cauliflower mosaic virus 35S promoter and tNos (Knoester et al., 1998). The resultant vector named HSVd-GFP reporter contains a monomeric modified HSVd cDNA (with an ATG insertion in the full-length HSVd-cDNA molecule) transcriptionally fused to the 5′ end of the GFP cDNA (Figure 1a). A binary pMog800 plasmid carrying the construction 35S:GFP:t-Nos (Sanchez-Navarro et al., 2006) was used for agroinfiltration and GFP expression control (Figure 1a).
Transgenic N. benthamiana
The HSVd-transgenic N. benthamiana lines HSVd2x-2 and HSVd2x-4 expressing and processing dimeric (+) HSVd-cDNA has been previously described (Gómez and Pallás, 2006). In all experiments the same plant material was used to determine the levels of low and high molecular weight RNAs, as well as protein expression by GFP fluorescence and western blot.
Binary pMog800 plasmids carrying the 35S-HSVd-GFP-tNos or 35S:GFP:tNos cassettes were transformed into Agrobacterium tumefaciens strain C58C1 containing the virulence helper plasmid pCH32 (Hamilton et al., 1996). The N. benthamiana HSVd-transgenic plants were agroinfiltrated as previously described (Vlot et al., 2001) in two basal leaves, and were maintained in environmentally controlled growing chambers (28°C, 14 h of light). After agroinfiltration (3 dpi), leaf discs (three per leaf) of approximately 5.5 mm in diameter were obtained. GFP expression in plants was analyzed with a Leica TCS SL confocal laser scanning microscope (Leica, http://www.leica.com), with excitation at 488 nm and emission at 510–560 nm.
RNA extraction and northern blot analysis
Total RNA were extracted using TRI reagent (Sigma, http://www.sigmaaldrich.com) according to the manufacturer’s instructions. Briefly, 250 mg of leaves from transgenic and control plants were ground in 2 ml of TRI reagent, 400 μl of chloroform was then added and the sample was vigorously vortexed, followed by centrifugation at 15 000 g. The supernatant was recovered and the total RNAs were precipitated with isopropanol. The RNA pellet was resuspended in sterile water and phenol-chloroform extracted. The supernatant was recovered and total RNAs were precipitated (1/10 volume 3 m sodium acetate, with two volumes ethanol) and resuspended in 50 μl of sterile water. The total RNA preparations were quantified by spectrometry, and their concentrations made equal.
To detect the small RNAs, 50 μg of total RNA was loaded on 15% polyacrylamide gel, 0.25 X TBE (90 mm Tris, 90 mm boric acid, 2 mm EDTA) and 8 m urea. The gel was run at 100 V for 2 h and then the RNA was transferred to positively charged Nylon membranes (Roche, http://www.roche.com). Hybridization was performed at 35°C for 14–16 h, using a Digoxigenin-labeled negative strand-specific RNA, obtained by transcription of plasmids pBdHSVd (Astruc et al., 1996), as a probe. The membrane was washed with 2X SSC, 0.1% SDS for 15 min at 35°C and 0.1X SSC, 0.1% SDS for 15 min at 37°C. Chemiluminescent detection was performed as previously described (Astruc et al., 1996).
To analyze the circular and linear forms of HSVd-RNA by northern blot analysis, 1.5 μg of the total RNA preparation was electrophoresed under denaturing conditions in a 5% polyacrylamide mini-gel, 0.25X TBE and 8 m urea (Pallás et al., 1987). After electrophoresis, the RNA was blotted to positively charged Nylon membranes and hybridized as previously described (Pallás et al., 1987).
Western blot assays
Total proteins were extracted by grinding four leaf discs (5.5 mm in diameter) in 100 μl of protein extraction buffer (150 mm Tris, pH 8.8, 5% SDS, 15% glycerol, 100 mm DTT). The homogenate was centrifuged for 1 min at 15 000 g in a microcentrifuge and the supernatant collected. The supernatant (30 μl) was denatured and fractionated by 10% SDS-PAGE and transferred to Hybond™-P membranes (GE Healthcare Life Sciences, http://www.gelifesciences.com) (Gómez et al., 2005). Membranes were blocked for 1 h [TBS (500 mm NaCl, 20 mm Tris, pH 7.5), 5% defatted milk, 2% BSA, 0.3% Tween 20] and incubated overnight at 4°C with an anti-GFP) mouse IgG monoclonal antibody (Roche). Membranes were washed (TBS, 0.3% Tween 20), incubated with ECL™ Peroxidase labeled anti-mouse antibody, and revealed by luminescence (ECL+Plus; GE Healthcare Life Sciences) according to the manufacturer’s instructions.
Two week-old N. benthamiana wild-type scions were grafted onto transgenic N. benthamiana stocks, as previously described (Gómez and Pallás, 2006). Grafted plants were kept for 5 days at room temperature (25°C) under controlled humidity conditions and were later moved to environmentally controlled growing chambers at 28°C. Total RNA were extracted from the scion leaves from 30-day-old grafted plants and analyzed by northern blot as described above.
We are indebted to Dr M.D. Gómez-Jimenez for excellent technical assistance in the observation and photography of GFP expression with the confocal microscope. We thank Drs R. Flores, C. Llave, J.A. Daròs, and S.F. Elena for their valuable contribution in the critical reading of the manuscript. This work was supported by grant BIO2005-07331 from the Spanish granting agency DGICYT and by grant GV05-238 from the Generalitat Valenciana. GG is the recipient of a contract from the CSIC.