Small RNAs containing the pathogenic determinant of a chloroplast-replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing
Article first published online: 4 APR 2012
© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd
The Plant Journal
Volume 70, Issue 6, pages 991–1003, June 2012
How to Cite
Navarro, B., Gisel, A., Rodio, M. E., Delgado, S., Flores, R. and Di Serio, F. (2012), Small RNAs containing the pathogenic determinant of a chloroplast-replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing. The Plant Journal, 70: 991–1003. doi: 10.1111/j.1365-313X.2012.04940.x
- Issue published online: 12 JUN 2012
- Article first published online: 4 APR 2012
- Accepted manuscript online: 14 FEB 2012 10:34AM EST
- Received 27 January 2012; accepted 9 February 2012; published online 2 April 2012.
- chloroplast development;
- heat-shock protein 90;
- non-coding RNAs;
- RNA silencing;
- viroid pathogenesis
- Top of page
- Experimental Procedures
- Supporting Information
How viroids, tiny non-protein-coding RNAs (∼250–400 nt), incite disease is unclear. One hypothesis is that viroid-derived small RNAs (vd-sRNAs; 21–24 nt) resulting from the host defensive response, via RNA silencing, may target for cleavage cell mRNAs and trigger a signal cascade, eventually leading to symptoms. Peach latent mosaic viroid (PLMVd), a chloroplast-replicating viroid, is particularly appropriate to tackle this question because it induces an albinism (peach calico, PC) strictly associated with variants containing a specific 12–14-nt hairpin insertion. By dissecting albino and green leaf sectors of Prunus persica (peach) seedlings inoculated with PLMVd natural and artificial variants, and cloning their progeny, we have established that the hairpin insertion sequence is involved in PC. Furthermore, using deep sequencing, semi-quantitative RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends (RACE), we have determined that two PLMVd-sRNAs containing the PC-associated insertion (PC-sRNA8a and PC-sRNA8b) target for cleavage the mRNA encoding the chloroplastic heat-shock protein 90 (cHSP90), thus implicating RNA silencing in the modulation of host gene expression by a viroid. Chloroplast malformations previously reported in PC-expressing tissues are consistent with the downregulation of cHSP90, which participates in chloroplast biogenesis and plastid-to-nucleus signal transduction in Arabidopsis. Besides PC-sRNA8a and PC-sRNA8b, both deriving from the less-abundant PLMVd (−) strand, we have identified other PLMVd-sRNAs potentially targeting peach mRNAs. These results also suggest that sRNAs derived from other PLMVd regions may downregulate additional peach genes, ultimately resulting in other symptoms or in a more favorable host environment for viroid infection.
- Top of page
- Experimental Procedures
- Supporting Information
Viroids are minimal RNAs that infect and often cause severe diseases in plants (Flores et al., 2005; Tsagris et al., 2008; Ding, 2009). Based on their properties, among which the ability to replicate through specific pathways of a rolling-circle mechanism in certain subcellular compartments is key, viroid species are classified into two families. The family Pospiviroidae, type species Potato spindle tuber viroid (PSTVd) (Diener, 1972; Gross et al., 1978), clusters viroids replicating and accumulating in the nucleus, whereas the family Avsunviroidae, type species Avocado sunblotch viroid (ASBVd) (Symons, 1981; Hutchins et al., 1986), includes viroids with hammerhead ribozymes in both polarity strands that mediate self-cleavage of their replicative intermediates generated in plastids (mostly chloroplasts) (Flores et al., 2000). Despite being composed of just a small (246–401 nt), circular, non-protein-coding RNA, viroids can usurp and redirect the host machinery for completing their infectious cycle. Therefore, viroids largely differ from viruses, the replication, movement and pathogenesis of which partly rely on proteins encoded in their own genome. However, similarly to changes incited by viruses, viroid infections cause profound changes in their host homeostasis (Itaya et al., 2002; Tessitori et al., 2007; Wang et al., 2011), which ultimately result in the onset of visible symptoms.
RNA silencing, a regulatory network that modulates host gene expression and protects plants and most other eukaryotes against invading nucleic acids, such as transposons, viruses and transgenes, is triggered by double-stranded RNAs (dsRNAs) and highly-structured single-stranded RNAs (ssRNAs) that are processed by Dicer-like (DCL) RNases into small RNAs (sRNAs) (Carthew and Sontheimer, 2009; Chen, 2009). The two major classes of sRNAs are microRNAs (miRNAs) and small interfering RNAs (siRNAs), with host RNA-dependent RNA polymerases (RDRs) mediating an amplification circuit resulting into secondary siRNAs able to promote RNA silencing in a non-cell-autonomous manner (Voinnet, 2008; Dunoyer and Voinnet, 2009). The sRNAs are loaded into AGO proteins and guide the RNA-inducing silencing complex (RISC) to target their cognate RNAs or DNAs (Mallory and Vaucheret, 2010).
To defend themselves, plant viruses encode in their genomes proteins suppressing the RNA-silencing response that they trigger in their hosts (Csorba et al., 2009; Ding, 2010). As a side effect, these RNA-silencing suppressors (RSSs) may impair host developmental pathways regulated by RNA silencing, explaining at least in part the phenotypic alterations accompanying most virus infections (Kasschau et al., 2003; Jay et al., 2011). However, in the absence of viroid-encoded RSSs, this model cannot account for symptoms induced by viroids. In addition to the conventional model regarding a direct interaction of the genomic viroid RNA with host proteins and/or RNAs as the primary event of viroid pathogenesis (reviewed by Flores et al., 2005), an intriguing alternative model was first proposed when viroid-derived sRNAs (vd-sRNAs), structurally similar to sRNAs, were detected in PSTVd-infected tissues (Papaefthimiou et al., 2001). Accordingly, symptoms would result from vd-sRNAs loading RISC and targeting specific host mRNAs for inactivation. This hypothesis has been extended to explain pathogenesis of satellite RNAs of plant viruses (Wang et al., 2004) and to involve RDR6-derived secondary siRNAs in viroid pathogenesis (Gómez et al., 2008).
Recently, direct experimental evidence has been supplied showing that the yellow symptoms induced in tobacco by the concurrent infection of Cucumber mosaic virus (CMV) and its Y-satellite RNA (Y-sat) result from silencing the chlorophyll biosynthetic gene CHLI by a 22-nt Ysat-derived siRNA (Shimura et al., 2011; Smith et al., 2011). However, in spite of multiple reports on the identification and characterization of vd-sRNAs accumulating in tissue infected by nucleus- and chloroplast-replicating viroids (Papaefthimiou et al., 2001; Martínez de Alba et al., 2002; Itaya et al., 2007; Di Serio et al., 2009, 2010; Navarro et al., 2009; St-Pierre et al., 2009; Bolduc et al., 2010; Martínez et al., 2010), attempts to validate this hypothesis for viroids have failed so far, with the evidence being circumstantial and restricted to correlating symptom severity with vd-sRNA accumulation (Markarian et al., 2004; Wang et al., 2004, 2011; Matoušek et al., 2007; Gómez et al., 2008; Diermann et al., 2010). Moreover, in most instances a similar correlation also exists with the genomic viroid RNA, making it difficult to draw reliable inferences (Itaya et al., 2001; Carbonell et al., 2008; Di Serio et al., 2010).
To address the issue of whether vd-sRNAs have a role in pathogenesis, Peach latent mosaic viroid (PLMVd) (Hernández and Flores, 1992) seems particularly convenient. PLMVd, a member of the family Avsunviroidae that replicates in the chloroplast (Bussière et al., 1999; Rodio et al., 2007), may induce in its natural host (Prunus persica, peach) a broad variety of symptoms that includes a severe albinism (peach calico, PC) in leaves, stems and fruits. PC is associated with variants of 348–351 nt containing a specific insertion of 12–14 nt (Malfitano et al., 2003) that is absent in latent and mosaic-inducing variants of 335–338 nt (Ambrós et al., 1998, 1999; Flores et al., 2006).
Interestingly, the pathogenic determinant for PC has been mapped at this insertion, which folds into a hairpin capped by a UUUU loop (Malfitano et al., 2003; Rodio et al., 2006). Further dissection of the molecular pathway underlying PC has shown that PLMVd variants inducing this syndrome interfere with the maturation of plastid rRNAs, thus impairing plastid translation and chloroplast biogenesis (Rodio et al., 2007). The recent release of the complete sequence of the peach genome (peach v1.0; International Peach Genome Initiative, http://www.rosaceae.org/peach/genome), provides a critical resource for genome-wide analyses of PLMVd-induced alterations in this host. Here, we report that the sequence of the pathogenic determinant for PC is crucial for eliciting this symptomatology, and supply direct evidence supporting that vd-sRNAs containing the PC determinant program RISC for cleaving a host mRNA, a finding with potential implications on viroid pathogenesis.
- Top of page
- Experimental Procedures
- Supporting Information
Molecular dissection of the pathogenic determinant for PC
In a previous work we showed that, in addition to the capping UUUU loop, the stem of the hairpin insertion seems to play a role in PC (Figure 1a) (Rodio et al., 2006). To further investigate this role, we selected two natural PLMVd variants with the same capping UUUU loop but differing stems: the reference PC-inducing variant C40 with a 4-bp stem (Malfitano et al., 2003) (Figure 1b, left), and variant P1.148 with a 3-bp stem that is unable to elicit this syndrome (Rodio et al., 2006) (Figure 1c, left). Next, the C and A at position 349 in variants C40 and P1.148, respectively, were changed into A and C to generate the artificial variants C40(A349) and P1.148(C349), in which the predicted stem of the hairpin insertion was shorter and longer than in their respective wild-type counterparts (Figure 1b–e, left panels). Slash inoculation of dimeric transcripts from the natural and mutated C40 and P1.148 variants, and dot-blot hybridization of leaf RNA preparations, showed that the four variants were highly infectious (the eight GF-305 peach seedlings of each block became infected) and generated similar viroid titers (data not shown). These plants were observed for symptom expression and, to establish a relationship between the structure and pathogenicity of the infecting variants, their progeny were cloned and sequenced 6 months post inoculation (6 mpi). To better define this relationship, the albino sectors of symptomatic leaves were carefully separated from the adjacent green tissues before extraction, as in a previous study (Rodio et al., 2006). In addition, for getting a more complete view of variant distribution within the PC-expressing seedlings, leaves from an asymptomatic (green) branch were also analyzed.
As anticipated, variant C40 elicited severe PC symptoms in the eight inoculated seedlings, whereas variant P1.148 did not cause any leaf symptom (Figure 1b,c). In line with earlier results (Malfitano et al., 2003; Rodio et al., 2006), most C40 progeny variants accumulating in albino tissues presented a minor modification (a G→A substitution, not disrupting the hairpin stem) (Figure 1b), whereas in two of the eight sequenced variants additional minor modifications (including a covariation and a substitution) enlarged the stem up to 5 bp (Figure 1b). Intriguingly, all PLMVd variants recovered from the asymptomatic branch of the same plant contained a similar extended 5-bp hairpin stem (Figure 1b, right panel). On the other hand, half of P1.148 progeny variants preserved the hairpin folding of the parental insertion, but the other half showed longer or shorter stems (Figure 1c).
Unexpectedly, all seedlings inoculated with the mutant variant C40(A349), and six of eight seedlings inoculated with the mutant variant P1.148(C349), also displayed PC (Figure 1d,e). However, the two parental variants were not recovered in their respective progeny. Instead, in both cases, the hairpin insertions of variants accumulating in albino and green tissues were similar, respectively, to those of variants from albino and green tissues of seedlings inoculated with their corresponding natural variants (Figure 1, compare panels d and e with b and c).
Altogether, results from these bioassays show that the typical hairpin insertion (composed of a UUUU loop and a 4-bp stem previously associated with the pathogenic determinant for PC) was prevalent in PLMVd progeny variants from albino tissues, irrespective of the specific hairpin insertion of the inoculated natural or mutant variant. In contrast, and also irrespective of the specific hairpin insertion of the inoculated variant, PLMVd progeny from green tissues mostly presented a hairpin insertion with a UUUU loop but with a 5-bp stem resulting from an additional A:U pair. These data strongly suggested that the latter variants did not incite PC. To provide direct support for this hypothesis, we inoculated eight GF-305 peach seedlings with a dimeric transcript of variant C40(A349)-g12, recovered from the progeny of variant C40(A349) in the previous experiment, and with an additional A:U pair in the 5-bp stem (Figure 1d, right panel). All inoculated seedlings remained asymptomatic and most progeny variants presented the same hairpin insertion (Figure S1), thereby showing that variants of this kind cannot elicit PC.
Collectively, these data support the view that the structural requirements conferring PC depend not only on the capping loop (UUUU) of the hairpin insertion, but also on the stem composition (Rodio et al., 2006). However, at this stage we could not clearly discern whether the sequence or the specific morphology of the hairpin insertion were required for inducing PC.
The primary rather than the secondary structure of the pathogenic determinant plays a major role in PC
We showed previously that the insertion found in some PLMVd variants always adopts a hairpin folding (Malfitano et al., 2003; Rodio et al., 2006). To examine whether the sequence of this hairpin also plays a role in PC, we generated the artificial variant C40(stem), which differs from its parental C40 in having the eight nucleotides forming the stem interchanged (Figure 2). Therefore, the sequence of the 12-nt hairpin insertion of variants C40(stem) and C40 are largely different, whereas their secondary structure is preserved.
Then, dimeric transcripts of these two variants were slash-inoculated in two blocks of eight peach seedlings each. Dot-blot hybridization showed that all inoculated plants were infected at 6 mpi, and that their viroid titer was similar (data not shown). Remarkably, plants inoculated with variant C40(stem) remained asymptomatic, with all progeny variants preserving the stem stability and some displaying mutations in the capping loop (Figure 2). In contrast, control seedlings inoculated with variant C40 expressed typical PC symptoms. Therefore, these data show that preservation of the secondary structure (folding into a hairpin with a capping UUUU loop) is not sufficient for conferring PC pathogenicity to this structural element, and strongly support that its primary structure, particularly the 12-nt sequence GA(A/G)CUUUUGUUC (hereafter named PC-associated insertion) characteristic of variants from albino tissues, is critical in this respect.
PLMVd-sRNAs derived from the pathogenic determinant for PC accumulate during infection
In view of the apparent role played by the sequence of the PC-associated insertion, we examined whether pathogenesis could operate by the silencing-based model proposed previously for viroids and plant satellite RNAs (Wang et al., 2004). Adapting this model to our system implicitly entails that (+) or (−) PLMVd-sRNAs involved in PC should contain, at least partially, the PC-associated insertion.
To explore this possibility we subjected the sRNAs accumulating in PC-expressing seedlings to deep sequencing. This approach has already been adopted in a study aimed at comparing the vd-sRNAs accumulating in leaves of GF-305 peach seedlings infected with the PC-inducing variant C40 and a mosaic-inducing PLMVd variant (GDS6) (Di Serio et al., 2009). This previous C40 sRNAs library was prepared from symptomatic leaves that also contained green sectors. Considering the role of the PC-associated insertion in pathogenesis and the uneven distribution of variants with this structural element in symptomatic plants (see above), we generated a C40 sRNA library from albino leaf tissues carefully separated from the surrounding green sectors in order to enrich it with vd-sRNAs from variants containing the PC-associated insertion. In parallel, we also generated an sRNAs library from leaves of a mock-inoculated peach seedling as a negative control. Gel-purified sRNAs were linked to bar-coded adaptors for deep sequencing of both libraries in the same channel.
Of approximately 20 815 300 reads, 96.2% were attributable to the two bar-coded sRNA preparations (53.2 and 46.8% for the C40 and the mock-inoculated sample, respectively). Reads for sRNAs between 18 and 26 nt (9 014 628 and 7 557 838 for the C40 and the mock-inoculated sample, respectively), adopted a profile with two prominent 21- and 24-nt peaks in both cases (Figure S2). When PLMVd-sRNAs matching perfectly the (+) and (−) sequences of variant C40 and its progeny (Figure 1b) were searched, 1 925 916 reads (21.3% of the total generated by sRNAs between 18 and 26 nt) were retrieved from the C40 sample, corresponding to 8015 non-redundant sequences (Appendix S1), with minimal PLMVd-related sequences detected in the mock-inoculated sample (3961 reads representing 0.05% of the total, 611 of which were non-redundant); the latter reads most likely result from a minor contamination, because none of these PLMVd-related sequences matched perfectly with the peach genome. In line with results of the previous deep-sequencing experiment (Di Serio et al., 2009), (+) PLMVd-sRNAs (57.6%) were slightly more abundant than their (−) counterparts (42.4%), with a similar polarity distribution being observed after segregating PLMVd-sRNAs in size classes (Figure S3). Moreover, also in line with previous results (Di Serio et al., 2009), size classes of 21 and 22 nt were largely prevalent (64.5 and 23.8%, respectively) within the PLMVd-sRNAs population, whereas a minor fraction of 20-, 23- and 24-nt vd-sRNAs (around 10.2% altogether) was retrieved (Figure S3). Mapping the PLMVds-RNA reads along the genomic (+) and (−) RNAs revealed specific hot-spot profiles (Figure S4). A parallel mapping of the non-redundant PLMVd-sRNAs also revealed profiles with hot spots corresponding, as expected, to genomic regions with high nucleotide variability in the progeny variants (Figure S5).
A search for PLMVd-sRNAs spanning totally or partially (in at least one nucleotide) the sequence of the PC-associated insertion from C40 and its progeny variants revealed 111 non-redundant (+) and (−) 21-nt RNAs (6993 reads). Additionally, 100 non-redundant (+) and (−) vd-sRNAs of 22 nt (6799 reads) were mapped at the same structural element. These data show that the PC-associated insertion is indeed a source of vd-sRNAs (hereafter referred to as PC-sRNAs), although at this stage we did not know whether, like genuine miRNAs, they might target host mRNAs for inactivation.
Host mRNAs are potentially targeted by PC-sRNAs
To search transcripts that could be targeted by PLMVd-sRNAs for RISC-mediated degradation, we took advantage of the complete sequence of the peach genome released recently (peach v1.0; International Peach Genome Initiative, http://www.rosaceae.org/peach/genome), and applied the RNAhybrid program (Rehmsmeier et al., 2004). The search was restricted to sRNAs of 21 nt because this is the prevalent size of PLMVd-sRNAs and plant miRNAs (Di Serio et al., 2009; St-Pierre et al., 2009; Figure S3). Moreover, we took into consideration that miRNA-directed mRNA cleavage in plants requires a high degree of base pairing, and that mismatches in bona fide miRNA:target duplexes are mostly located in the first position and after position 13 relative to the 5′ terminus of the miRNA (Mallory et al., 2004). Therefore, a position-dependent scoring matrix for predicting plant miRNA targets (Fahlgren and Carrington, 2010) was applied to all possible duplexes formed by 21-nt PLMVd-sRNAs and predicted peach mRNAs identified as potential targets. A maximum score cut-off of 2.5, which is below the cut-off of 3.5 established for reliable sensitivity and specificity in Arabidopsis thaliana (Fahlgren and Carrington, 2010), was adopted. This cut-off of 2.5 seemed appropriate for our search because the duplexes between miRNAs identified in our libraries conserved in other species (including miR156, miR159, miR160, miR162, miR164, miR166, miR168, miR171, miR172, miR393, miR394, miR395, miR396 and miR397) and their potential targets in the complete peach genome showed scores from 0 to 3. The search resulted in 65 predicted peach mRNA targets, most encoding proteins with functional annotation (P. persica– JGI v1.0; http://www.phytozome.net/cgi-bin/gbrowse/peach; Table S1), and 30 of them having been detected as peach transcripts (peach assembled expressed sequence tags, ESTs, from peachv1pasa_assemblies).
Considering that the sequence of the PC-associated insertion is critical for eliciting PC, and that this insertion is a source of vd-sRNAs (PC-sRNAs) in albino tissues (see above), we next concentrated on the duplexes formed by PC-sRNAs and peach mRNAs detected as transcripts (Table 1). Overall, 12 PC-sRNAs were found potentially targeting 10 predicted peach mRNAs because PC-sRNA1 and PC-sRNA6 could target two different mRNAs (Table 1). This table also shows that two of the ten encoded proteins contain a predicted chloroplastic transit peptide (cTP) (Li and Chiu, 2010), according to chloroP software (http://www.cbs.dtu.dk/services/ChloroP) (Emanuelsson et al., 1999), thus indicating that PC-sRNAs might impair the expression of nuclear genes coding for plastid proteins. Nine and three PC-sRNAs were of (+) and (−) polarity, respectively (Table 1). Altogether, these data show that PC-sRNAs potentially targeting peach mRNAs are actually generated in albino tissues, opening the possibility that at least some could act like endogenous miRNAs or siRNAs.
The mRNA encoding the chloroplastic heat shock protein 90 (cHSP90) is targeted by PC-sRNAs for sequence-specific degradation
To provide direct evidence for the sequence-specific degradation of a peach mRNA mediated by PC-sRNAs, we focused on peach transcript ppa001590m for several reasons. First, two PC-sRNAs (PC-sRNA8a and PC-sRNA8b) were identified potentially targeting this transcript for cleavage, with the corresponding duplexes showing scores ranging among the lowest recorded (Table 1). Second, PC-sRNA8a and PC-sRNA8b, which derive from the viroid (−) strand, completely span the PC-associated insertion. And third, ppa001590m transcript codes for a protein of 797 amino acids annotated as homologous with CR88 of A. thaliana (84.8% similarity with AT2G04030.1), a chloroplast-targeted HSP90 protein (cHSP90) (Cao et al., 2003). A multiple alignment using clustalw (Thompson et al., 1994) showed that peach cHSP90 also displays high similarity with cHSP90 proteins from other plant species and Chlamydomonas reinhardtii (Figure 3), including the four domains conserved in the HSP90 family (Meyer et al., 2003), and the DPW C-terminal motif exclusively found in the plastidic members (Chen et al., 2006). Moreover, within the N-terminal 67 amino acids of peach cHSP90, the chloroP software predicted a cTP with a length and score similar to those obtained for the homologous proteins from the other species (Table S2). Therefore, these data strongly suggest that peach cHSP90 is a nuclear-encoded protein with plastidic localization. Interestingly, PC-sRNA8a and PC-sRNA8b target the sequence coding for the cTP (Figure 3). Furthermore, peach cHSP90 appears particularly appealing in the context of PC pathogenesis because its homologous CR88 is required for chloroplast biogenesis in A. thaliana (Cao et al., 2003); this protein, perhaps interacting with HSP70, may be needed for assembling the core of a multichaperone complex involved in the biogenesis/maintenance of thylakoid membranes (Schroda and Mühlhaus, 2009), which is the developmental pathway specifically altered in PC-expressing tissues (Rodio et al., 2007).
We next proceeded comparing the steady-state level of cHSP90 mRNA in mock-inoculated and PLMVd-infected peach seedlings expressing PC. In line with data from the peach EST database, RT-PCR amplification of a 405-bp cDNA corresponding to the 5′ terminus of the peach mRNA for cHSP90, followed by cloning and sequencing, showed that the gene cHSP90 is indeed transcribed in the mock-inoculated control. However, RT-PCR estimates using the same primers and serial cDNA dilutions showed a substantially lower steady-state level of the cHSP90 transcript in albino leaf sectors than in green tissues [from the same C40-inoculated seedling, or from a mock-inoculated seedling or a seedling infected by the latent variant C40(A349)-g12]. As additional controls we also tested the levels of the transcripts from genes psbA and rpoB, which were lower and higher in albino than in green tissues, respectively (Figure 4), in agreement with a previous report showing that mRNAs from genes transcribed by the plastid-encoded polymerase (like psbA) are negatively affected in albino tissues, whereas those transcribed by the nuclear-encoded polymerase (like rpoB) over-accumulate (Rodio et al., 2007).
The lower accumulation of the cHSP90 transcript in albino tissues is consistent with its targeting by PC-sRNA8a and PC-sRNA8b for RISC-mediated degradation (Figure 5). This hypothesis was validated by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) experiments with RNA preparations from albino tissues: four out of five cDNA clones were from fragments of the cHSP90 transcript with 5′ termini identical to the cleavage site predicted by PC-sRNA8a and PC-sRNA8b (Figure 5), between positions 10 and 11 from their 5′ termini, with the cleavage site inferred from the other clone mapping at one nucleotide downstream. These data conclusively showed that PC-sRNA8a and PC-sRNA8b downregulate a peach mRNA, as predicted by an RNA silencing mechanism. Conversely, the lack of the same RLM-RACE products when using RNA preparations from green tissues [from the C40-inoculated seedling, or from a mock-inoculated seedling or a seedling infected by the latent variant C40(A349)-g12], together with the similar cHSP90 mRNA levels detected in the same tissues (Figure 4), indicate that this peach transcript is not targeted for RISC-mediated degradation in the asymptomatic tissues. Finally, these findings are also consistent with the biased distribution of PLMVd variants observed in symptomatic seedlings (Figure 1): PCsRNA8a and PC-sRNA8b are exclusively generated from variants containing the PC-associated insertion, which only accumulate in albino leaf sectors. To give further credence to this view, two additional peach sRNA libraries were generated and sequenced from green leaves of PC-expressing and non-symptomatic seedlings inoculated with variants C40 and P1.148, respectively (Figure S6). Although PLMVd-sRNAs deriving from the hairpin insertion were sequenced in both libraries, none contained the PC-associated insertion. In particular, PC-sRNA8a and PC-sRNA8b were not found, and the PLMVd-sRNAs sequenced that could form duplexes with cHSP90 mRNA were derived from insertions of variants characteristically accumulating in green tissues. However, the corresponding scores were high (from 5 to 7.5; see Figure S7), further supporting the hypothesis that the sRNAs resulting from PLMVd variants without the PC-associated insertion do not prime RISC for cHSP90 mRNA cleavage.
- Top of page
- Experimental Procedures
- Supporting Information
In the first part of this study we furthered the characterization of the PLMVd determinant for PC by dissecting albino and green sectors of GF-305 peach seedlings inoculated with natural and artificial variants of this viroid, and subsequently cloning the resulting progeny. This approach has revealed the 12-nt sequence GA(A/G)CUUUUGUUC as the insertion conferring PC to infecting PLMVd variants. In agreement with previous data (Malfitano et al., 2003; Rodio et al., 2006), this insertion folds into a hairpin capped by a UUUU loop, and differs in the stem composition from other hairpin insertions found in PLMVd variants recovered from green tissues. Therefore, the primary rather than secondary structure of the pathogenic determinant is involved in eliciting PC, as clearly illustrated by the artificial variant C40(stem). The infecting ability of this variant probably results from the eight mutations introduced not affecting the stem stability of the hairpin insertion (Figure 2), in line with previous observations with another member of the same genus, Chrysanthemum chlorotic mottle viroid (CChMVd; Navarro and Flores, 1997), which also remains viable as long as the introduced mutations preserve elements of its secondary and tertiary structure (De la Peña et al., 1999; Gago et al., 2005).
Altogether, these findings are consistent with the silencing-based model of pathogenesis proposed for viroid and/or satellite RNAs (Papaefthimiou et al., 2001; Wang et al., 2004), recently validated experimentally for the yellow symptoms induced by a specific satellite RNA (Y-sat) of CMV (Shimura et al., 2011; Smith et al., 2011). However, regarding viroids, previous attempts to obtain support for this model have met with limited success. Although some reports highlight the positive correlation between symptom severity and the accumulation of vd-sRNAs in different plant–viroid combinations (Itaya et al., 2001; Markarian et al., 2004; Matoušek et al., 2007), including transgenic Solanum lycopersicum (tomato) lines expressing a non-infectious PSTVd hairpin RNA (Wang et al., 2004), others have failed to find a similar correlation in some transgenic lines expressing the same non-infectious PSTVd hairpin RNA, and apparently accumulating similar vd-sRNA titers (Schwind et al., 2009). Moreover, although RDR6, an enzyme mediating synthesis of secondary siRNAs, has been involved in symptom induction by Hop stunt viroid in Nicotiana benthamiana (Gómez et al., 2008), symptoms elicited by PSTVd in the same plant species are RDR6-independent (Di Serio et al., 2010). Setting aside these contradictory results, none of the previous studies have provided direct experimental support for the vd-sRNAs actually mediating cleavage of host mRNAs; at best, a correlation between the downregulation of tomato mRNAs potentially targeted by PSTVd sRNAs and symptom expression has been found (Wang et al., 2011). Finally, even if vd-sRNAs may target and downregulate, in a sequence-specific manner, the overexpression of a reporter gene (Vogt et al., 2004; Itaya et al., 2007) or the accumulation of viroid RNA in infected plants (Carbonell et al., 2008), these results do not prove that a similar mechanism operates against host genes in natural infections.
In the second part of this study we supply solid experimental evidence showing that the accumulation level of the mRNA coding for the chloroplast-targeted cHSP90 from peach is reduced in the albino sectors of PC-expressing leaves. Our data, obtained by a combination of deep sequencing, semi-quantitative RT-PCR and RLM-RACE, are consistent with the sequence-specific cleavage of this mRNA guided by PLMVd-sRNAs (PC-sRNA8a and PC-sRNA8b) generated from the PC-associated insertion of some PLMVd variants, thus illustrating that at least this viroid can indeed modify its host gene expression through RNA silencing. Chloroplast developmental defects and malfunctioning of plastid-to-nucleus signaling reported in PC-expressing tissues (Rodio et al., 2007) support a role for the downregulation of cHSP90 in eliciting the albino phenotype, because this chloroplast-targeted protein has been involved in chloroplast biogenesis and plastid-to-nucleus signal transduction in Arabidopsis and Chlamydomonas (Cao et al., 2003; Willmund and Schroda, 2005; Willmund et al., 2008). Additional support for this view comes from the yellow phenotype observed in a mutant line of A. thaliana with a single amino acid change in cHSP90 (Cao et al., 2003), and from two cHSP90 T-DNA null-mutant lines (EMB 1956-1 and EMB 1956-2) of A. thaliana that are embryo defective, and display white embryos and seeds (Meinke et al., 2008).
Although it has recently been shown that the yellow symptoms induced in Nicotiana tabacum by the Y-sat of CMV result from silencing the chlorophyll biosynthetic gene CHLI with a 22-nt siRNA derived from this satellite RNA (Shimura et al., 2011; Smith et al., 2011), another more complex RNA silencing-based model has been offered for explaining the attenuation of the yellowing symptomatology of N. benthamiana induced by a different satellite RNA of the same virus (Hou et al., 2011), indicating that the pathogenic mechanism proposed for Y-sat may not be general. Whether the silencing of gene cHSP90 guided by PC-sRNA8a and PC-sRNA8b is sufficient for inciting PC is not known because, at this stage, we cannot rule out the involvement of other factors. However, PLMVd-sRNAs other than PC-sRNAs can be excluded as direct players in this respect, because they lack nucleotides derived from the insertion strictly associated with PC.
Nine and three PC-sRNAs potentially targeting peach mRNAs have (+) and (−) polarity, respectively (Table 1), but both PC-sRNA8a and PC-sRNA8b derive from the (−) polarity strand. These results indicate that the activity of vd-sRNAs in promoting host mRNA cleavage may not depend on their relative abundance. In fact, the PLMVd region encompassing the PC determinant does not map at a hot spot in the profile of vd-sRNA reads (Figure S4); neither does the Y-sat region from which the 22-nt (+) species silencing CHLI mRNA derives (Shimura et al., 2011; Smith et al., 2011). This finding is in agreement with results of a previous study in which viroid dsRNAs, probably produced in the cytoplasm by host RDR(s), were proposed to serve as substrates for generating PLMVd-sRNAs (Di Serio et al., 2009). Consistent with this dsRNA-based origin of most PLMVd-sRNAs, the passenger strand of PC-sRNA8b was also identified in the sRNA library (Appendix S1, sequence ID: MAC-18-1596391_21_3_0), but with a number of reads (only three) significantly lower than that of its guide counterpart (169 reads; Table 1) mediating cleavage of the cHSP90 mRNA. Two other points are worthy of note: (i) PC-sRNA1 and PC-sRNA6 may form duplexes with more than one peach mRNA, suggesting that a small viroid region has the potential for silencing multiple host targets; and (ii) the U prevalence in the four 5′ terminal positions of PC-sRNA8a and PC-sRNA8b is remarkable because this trait is also found in the 22-nt siRNA from Y-sat of CMV silencing CHLI mRNA.
As PLMVd accumulates genetic heterogeneity very rapidly (Ambrós et al., 1999; Rodio et al., 2006; this work), in line with the observation that the related CChMVd has the highest mutation rate reported for any biological entity (Gago et al., 2009), new PLMVd-sRNAs may be generated with time, targeting other peach genes and eventually resulting in the fluctuating phenotype characteristic of most PLMVd infections (Flores et al., 2006). Consistent with this view, most PLMVd-sRNAs with potential peach targets derive from viroid regions displaying high sequence variability (Figure S5). As previously proposed for viruses (Moissiard and Voinnet, 2006; Llave, 2010), changes in host gene expression induced by PLMVd-sRNAs could generate a more favorable environment for viroid infection. Reciprocally, viroid modulation of host gene expression via RNA silencing might play a role in the molecular evolution of these minimal infectious agents.
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- Experimental Procedures
- Supporting Information
Plant material and growing conditions
Leaf tissues were from GF-305 peach [Prunus persica (L.) Batsch] seedlings slash-inoculated with buffer or dimeric head-to-tail transcripts generated in vitro from natural and artificial PLMVd variants inducing different foliar symptoms or no symptoms (see below). Six weeks after inoculation, symptom expression was stimulated by chilling the seedlings at 4°C in the darkness for 6–8 weeks, and then transferring them back to the glasshouse to favor the emergence of new flushes.
RNA extraction and dot-blot hybridization
Total nucleic acid preparations were extracted from leaves (60 mg) with phenol-chloroform, recovered by ethanol precipitation and resuspended in water (50 μl) (Rodio et al., 2007). When indicated, albino sectors from PC-expressing leaves were excised from the surrounding green tissues with a razor blade. PLMVd accumulation in infected tissues was quantified by spotting aliquots (5 μl) of 1/5, 1/25 and 1/125 dilutions of the nucleic acid preparations onto positively charged nylon membranes (Roche Diagnostics GmbH, http://www.roche.com) that were hybridized with a PLMVd-specific digoxigenin-labeled riboprobe (Rodio et al., 2006).
Amplification and sequencing of PLMVd progeny variants
For cloning PLMVd-cDNAs from progeny variants, nucleic acids preparations (150 μl) were obtained from leaf tissues (60 mg) by a modified silica-gel capture system (Foissac et al., 2001); when dealing with symptomatic leaves, albino and green sectors were dissected beforehand. Aliquots (5 μl) were used for synthesizing first-strand cDNA with random hexamers and the High Capacity Reverse Transcription kit (Applied Biosystem, http://www.appliedbiosystems.com). The resulting cDNAs were PCR-amplified with the primer pair FPLMV-57 (5′-CACACCCCCCTCGGAACCAACCG-3′) and FPLMV-58 (5′-ATCCAGGTACCGCCGTAGAAAC-3′), complementary and identical to positions 202–180 and 203–224 of the reference PLMVd variant (Hernández and Flores, 1992; Ambrós et al., 1998), respectively, and the Expand High Fidelity PCR system (Roche Diagnostics GmbH). Amplicons of the expected size were cloned in p-GEM-T Easy plasmid (Promega, http://www.promega.com) and sequenced (MWG-Biotech, http://www.mwg-biotech.com). The GenBank IDs for PLMVd progeny variants are: JN377825–JN377849; JN377851–JN377862; JN377864–JN377874; and JN377876–JN377891.
PLMVd infectious clones, site-directed mutagenesis and inoculation of in vitro transcripts
Plasmids containing head-to-tail PLMVd-cDNA dimeric inserts from natural variants C40 (AJ550912) and P1.148 (DQ222050) have been described previously (Malfitano et al., 2003; Rodio et al., 2006); for descriptions of plasmids of variants C40(A349)-g12 (JN377855), P1.148(C349) (JN377863), C40(A349) (JN377850) and C40(stem) (JN377875), see Appendix S2. Recombinant plasmids were linearized with appropriate restriction enzymes and transcribed with T7 or SP6 RNA polymerases. The resulting products were analyzed by electrophoresis in 5% polyacrylamide gels containing 1X TBE buffer and 8 m urea, eluted and slash-inoculated into GF-305 peach seedlings.
Deep sequencing and bioinformatics analyses
The protocol for purifying the sRNAs, adaptor ligation, RT-PCR amplification, library purification and high-throughput DNA sequencing on the Illumina Genome Analyzer (Fasteris SA, http://www.fasteris.com), has been reported (Di Serio et al., 2010). Four libraries were sequenced. Two bar-coded leaf samples, from mock-inoculated and C40-infected GF-305 seedlings, were analyzed in a single channel in the Illumina EAS269 GAII; the sRNA library from the C40-infected GF-305 seedling was generated from albino sectors dissected from adjacent green tissues. The two additional bar-coded libraries from green leaf tissues of C40- and P1.148-infected GF305 seedlings were sequenced in a single channel in the Illumina Genome Analyzer HiSeq 2000. Raw data from the Illumina platform were fed into an in-house pipeline, which removed barcodes and adaptors and split the clean sequences by size. Sequence sizes between 18 and 26 nt were blasted (BLASTN; Altschul et al., 1997) against a selected set of PLMVd variants, and a mapping profile against the consensus of the multiple alignment (clustalw; Thompson et al., 1994) was generated. To find out potential targets of PLMVd-sRNAs, sequences perfectly matching genomic RNAs of C40 and its progeny were used for RNAhybrid (Rehmsmeier et al., 2004) searching on exon sequence fragments of the peach genome (Peach v1.0; International Peach Genome Initiative, http://www.rosaceae.org/peach/genome). The quality of the duplex pairing was estimated as proposed previously (Fahlgren and Carrington, 2010). Peach mRNAs detected as transcripts and forming potential duplexes with PLMVd-sRNAs spanning totally or partially (in at least one nucleotide) the PC-associated insertion (PC-sRNAs) were further analysed by chloroP (http://www.cbs.dtu.dk/services/ChloroP/; Emanuelsson et al., 1999) to predict the presence of chloroplastic transit peptides (Li and Chiu, 2010) in the encoded proteins.
Transcript levels were estimated by RT-PCR. Total RNAs (200 ng), obtained by treating total nucleic acid preparations with RQ1 DNase I (Promega), were reverse transcribed with random hexamers. Aliquots (2 μl) of serial dilutions (1:2, 1:20 and 1:200) of the resulting cDNAs were added to amplification reactions (25 μl) catalyzed with Go-Taq DNA polymerase (Promega). The cycling program, consisting in an initial denaturation at 94°C for 3 min and 30 cycles (94°C for 30 s, 50°C for 30 s for 18S, psbA and RpoB, and 55°C for cHSP90, and 72°C for 30 s), with a final extension at 72°C for 7 min, was adopted according to preliminary experiments showing that cDNA amplification was in the logarithmic phase. A cDNA fragment (405 nt) of cHSP90 mRNA was RT-PCR amplified with primers cHSP90-Fw (5′-CAATGGCTCCAGTTCTAAGCA-3′) and cHSP90-Rv (5′-GCAGAGAGGGCTCAGTCACACTCAA-3′), identical and complementary, respectively, to positions 84–104 and 464–488 of this transcript. Sequencing of the resulting product confirmed the expected cDNA (JN377892). Primer pairs for cDNA amplification of the 18S rRNA and psbA and RpoB transcripts have been described previously (Rodio et al., 2007).
5′ RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE)
RQ1 DNase I-treated total RNAs (1.2 μg) were incubated at 20°C for 6 h with an RNA adaptor (5′-GUUCAGAGUUCUACAGUCCGACGAUC-3′) and 0.5 U of T4 RNA ligase (Promega). Ligated RNAs were reverse transcribed with primer cHSP90-Rv as described before. First PCR was then performed using the forward primer P2 (5′-AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3′), with the 3′ moiety (in bold) homologous to the RNA adaptor, and the reverse primer cHSP90-Rv reported above, whose 5′ end maps 288 nt downstream the cleavage site of peach cHSP90 mRNA predicted by PC-sRNA8a and PC-sRNA8b. The resulting product was amplified with the same primer P2 and the nested reverse primer cHSP90-nes-Rv (5′-CCTTGTGGCTGTATAGACTATG-3′), complementary to positions 383–404 of peach cHSP90 mRNA. Following electrophoresis in a 1.2% agarose gel, the nested PCR product (248 bp) was excised, cloned into the pGEM-T easy vector (Promega) and sequenced.
- Top of page
- Experimental Procedures
- Supporting Information
This work was supported by a dedicated grant from the Italian Ministry of Economy and Finance to the CNR (Legge n. 191/2009), the Dipartimento Agroalimentare of the CNR of Italy (A. Leone and D. Mariotti 2008 award for advanced research in agriculture to FDS) and by the Ministerio de Ciencia e Innovación of Spain (grants BFU2008-03154 and BFU2011-28443 to RF).
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- Experimental Procedures
- Supporting Information
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- Top of page
- Experimental Procedures
- Supporting Information
Figure S1. Peach phenotype and progeny of natural PLMVd variant C40(A349)-g12. The hairpin insertion of variant C40(A349)-g12 (with light blue background) and the flanking 5’ and 3’ nucleotides are depicted on the left. The sequence variability of the hairpin insertions (encircled nucleotides) and the name of the progeny variants recovered from green (g) tissues of one representative inoculated seedling are indicated. The ratio of symptomatic to infected seedlings appears between brackets on the top of the panel.
Figure S2. Relative distribution by size classes of total sRNAs from GF-305 peach leaves infected by variant C40 (blue) or mock-inoculated (red). The fraction (%) of each sRNA class of a given length has been plotted against its length.
Figure S3. Distribution by size classes, polarity and abundance of PLMVd-sRNAs in the C40-infected sample. Histograms show the total reads of (+) and (-) PLMVd-sRNAs, in green and gray, respectively, of a given length.
Figure S4. Location and frequency of the 5’ termini of PLMVd-sRNAs along the genomic RNAs. Bars above and below the x-axis represent (+) and (-) PLMVd-sRNAs reads, respectively. Note that the same numbering (referred to variant C40) is used in (+) polarity (5’ to 3’ orientation is from left to right) and in (-) polarity (5’ to 3’ orientation is from right to left). Blue and red horizontal lines denote, respectively, (+) and (-) PLMVd-sRNAs with at least one nucleotide mapping within the hairpin insertion. Bars with blue and red asterisks indicate that the corresponding reads (223401 and 86844) for (+) and (-) PLMVd-sRNAs with 5’ termini at positions 216 and 251, respectively, are out of the scale.
Figure S5. Number and location of the 5’ termini of 21-nt non-redundant (nr) PLMVd-sRNAs along the genomic RNAs (panel a) and nucleotide variability detected in variant C40 and its progeny (panel b). (a) Bars above and below the x-axis represent numbers of (+) and (-) nr-PLMVd-sRNAs, respectively. Note that the same numbering (referred to variant C40) is used in (+) polarity (5’ to 3’ orientation is from left to right) and in (-) polarity (5’ to 3’ orientation is from right to left) and that numbering starts at position 23 to avoid splitting the hairpin insertion. Red and green squares denote, respectively, (+) and (-) PLMVd-sRNAs with at least one nucleotide within the hairpin insertion. Horizontal arrows map (+) (in red) and (-) (in green) 21-nt PLMVd-sRNAs with potential targets in the peach genome. Arrowheads indicate the 5’-3’ orientation of the PLMVd-sRNAs. (b) y-axis, number of polymorphisms detected at each position of variant C40 and its progeny. Nucleotide insertions and deletions are denoted by upwards and downwards arrows, respectively. Note that most PLMVd-sRNAs with potential targets in the peach span regions with high number of nr-PLMVd-sRNAs and high sequence variability.
Figure S6. Distribution by size classes, polarity and abundance of PLMVd-sRNAs in the green tissue of C40- and P1.148-infected samples. Histograms show the total reads of (+) and (-) PLMVd-sRNAs, in green and gray, respectively, of a given length.
Figure S7. Duplexes potentially formed by the cHSP90 mRNA and PLMVd-sRNAs with hairpin insertions obtained by deep sequencing of sRNA libraries from green leaves of C40- and P1.148-infected peach seedlings (panels a and b, respectively). Nucleotides in bold correspond to the hairpin insertion. The scores of RNA duplexes are calculated as in Fahlgren and Carrington (2010), and the number of reads is indicated between brackets on the right.
Table S1. Predicted peach mRNAs targeted by PLMVd-sRNAs matching perfectly the genomic sequence of variant C40 and its progeny.
Table S2. Chloroplast transit peptides and their length in cHSP90 of five species predicted by the ChloroP program.
Appendix S1. PLMVd-sRNAs (18-26 nt) from albino leaf sectors infected with PLMVd variant C40.
Appendix S2. Experimental procedure. Construction of plasmids containing head-to-tail PLMVd-cDNA dimeric inserts.
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