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Received Date : 25-Feb-2014 Revised Date : 09-May-2014 Accepted Date : 21-May-2014 Article type

: Original Article

Virus induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two different patterns of transgene body methylation

Mingmin Zhao, David San León, Ma. Otilia Delgadillo, Juan Antonio García and Carmen Simón-Mateo*. Centro Nacional de Biotecnología (CSIC), Campus Universidad Autónoma de Madrid, 28049, Madrid, Spain.

Key words: Virus induced gene silencing, viral resistance, DNA methylation, posttranscriptional gene silencing, gene body methylation.

Running head: Two different DNA methylation patterns upon VIGS

* Corresponding author: Mailing address: Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049 Madrid, Spain. Phone: +34-915855397. Fax: +34-915854506. E-mail: [email protected]

SUMMARY We have studied by bisulphite sequencing the methylation of a viral transgene whose expression was silenced upon Plum pox virus infection of the transgenic plant and subsequent

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recovery as a consequence of the so-called virus-induced gene silencing (VIGS). VIGS was associated with a general increase in the accumulation of small RNAs corresponding to the coding region of the viral transgene. After VIGS the transgene promoter was not methylated and the coding region showed uneven methylation, the 5´ end region was mostly not methylated in the recovered tissue or mainly methylated in CG sites in regenerated silenced plants. The methylation increased towards the 3´ end region, which had a dense methylation in all three types of contexts (CG, CHG, and CHH). This methylation pattern and the corresponding silenced status were maintained after plant regeneration from recovered silenced tissue and did not spread into the promoter region, but were not inherited in the sexual offspring. Instead, a new pattern of methylation was observed in the progeny plants consisting of the disappearance of the CHH methylation, a remaining CHG methylation at the 3´ end and an overall increase of the CG methylation in the 5´ end. The latter epigenetic state was inherited over several generations and did not correlate with transgene silencing and accordingly virus resistance. These results suggest that the widespread CG methylation pattern found in body genes located in euchromatic regions of plant genomes could also reflect an older silencing event and most likely these genes are not longer silenced.

INTRODUCTION Methylation of cytosines in DNA is a common eukaryotic DNA modification identified as a powerful mechanism to regulate gene expression through gene silencing. It is essential for the normal development of most multicellular organisms and DNA methylation patterns need to be both stable, that is heritable, and flexible in time and space for normal development to occur. According to the sequence context of the methylated cytosines, DNA methylation can be categorized in three types depending on the sequence context: symmetric sites CG or CHG, or a non-symmetric context CHH (H=A, C or T). In mammals, methylated Cs are located almost exclusively within CG dinucleotides, whereas in plants, Cs in the symmetric triplet CHG and in non-symmetric sequence contexts are also methylated, although less efficiently than Cs in CGs. This difference in methylation patterning reflects the different types of cytosine methyltransferases controlled by different genetic pathways present in plants (Law and Jacobsen 2010; Meyer 2011). It is noteworthy that silencing of gene expression and DNA methylation pathways frequently co-exist. In the last few years abundant information has been gathered on the interplay between DNA methylation and promoter inactivation, the so-called transcriptional gene silencing (TGS) (Mette et al. 1999; Vaucheret and Fagard 2001). However genome-wide data on the methylome of different organisms have revealed that the methylation in the

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transcribed regions, specifically in exons, of endogenous genes is unexpectedly widespread (Feng et al. 2010). In Arabidopsis fewer than 5% of expressed genes were shown to have methylated promoters, however about one-third of genes were methylated in their open reading frame. Although DNA methylation of promoters is known to inhibit transcription initiation, the significance and effects of methylation within the gene body are not fully understood. In many cases the loss of body methylation does not seem to trigger a systematic and drastic overexpression of body-methylated genes to the same extent as transposon reactivation. However, a moderate up-regulation of body-methylated genes was observed, suggesting that body methylation might be involved in fine-tuning transcription levels (Lee et al. 2010; Zemach et al. 2010; Zilberman et al. 2007).

Interestingly, the first observation that DNA methylation led to stable gene silencing came from transgenesis in plants. In genetically modified plants, transgene constructs frequently undergo silencing. TGS takes place when the promoter is inactivated, while in posttranscriptional gene silencing (PTGS) transcription takes place but the primary transcript is rapidly degraded. Both types of silencing are associated with de novo methylation of cognate sequences, DNA methylation in TGS occurs in the promoter region (Mette et al. 2000; Zhang et al. 2006); and in PTGS is associated with DNA methylation in the coding sequences (Dalakouras et al. 2012; Fojtová et al. 2006; Guo et al. 1999; Jones et al. 1998a; LunerováBedřichová et al. 2008; Vermeersch et al. 2013). Whereas it has been demonstrated that RNA can trigger promoter methylation that results in TGS, it still remains unclear the causal relationships between DNA methylation of transcribed sequences and PTGS. The induction of the methylation of the coding region of a transgene was first described during infection of plants with a viroid (Wassenegger et al. 1994). Similarly, transgene silencing can be induced upon infection with a virus carrying portions of host genes (virus induced gene silencing, VIGS) (Kumagai et al. 1995; Ruiz et al. 1998), which is sometimes associated with sequence-specific methylation (Guo et al. 1999; Jones et al. 1998b). However, there are also examples in which PTGS-associated methylation has not been observed (Wang and Waterhouse 2000). Although the initiation, maintaining, resetting and finally the precise role of gene body methylation in PTGS remain essentially unknown, it is clear that a good understanding of the mechanisms involved in this process has implications both for basic plant biology and for applied purposes. In the present study we have studied the DNA methylation of a transgene of the Nicotiana benthamiana line NIbV transformed with the NIb region of the potyvirus Plum pox virus (PPV) genome. In line NIbV the methylation and virus resistance occurs after PPV infection due to VIGS. The DNA methylation has been studied in detail using a sensitive method such as bisulfite sequencing to obtain detailed information on the methylation status of

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each cytosine in the transgene. This type of transgenic line is particularly suited to examine the consequences of RNA-triggered epigenetic changes induced by VIGS and to assess the inheritance of these changes.

RESULTS Methylation analysis of the transgene in recovered NIbV plants. Previous studies showed that transgenic N. benthamiana NIbV-3 line (NIbV), transformed with a mutated NIb coding sequence from Plum pox virus (PPV), showed a delayed, very specific, resistance phenotype, which was induced upon infection (Guo and García 1997). This recovery was the consequence of the activation in the PPV-infected plant of an RNA silencing mechanism derived from virus infection (virus-induced gene silencing, VIGS). As in other examples, in line NIbV DNA methylation of the transgene was associated with establishment and maintenance of transgene silencing and virus resistance. The recovery phenotype was not meiotically stable and the progeny plants coming from selfing of a recovered plant have normal transgene mRNA levels and initial susceptibility to PPV infection (Guo et al. 1999). Nevertheless, plantlets regenerated in vitro from asymptomatic leaf explants of initially infected plants (REG) could maintain both transgene silencing and resistance to PPV in the absence of the inducer PPV infection (Guo et al. 1999; Simón-Mateo et al. 2003, and Fig. 1A and B).

In order to obtain detailed data on the methylation status of each cytosine of the transgene in silenced tissue we used bisulfite sequencing which allows the detection of all methylated cytosine residues (Cs) within a DNA region. The primer combinations used in these analyses were designed to allow amplification of methylated, partially methylated and non-methylated copies of the transgene in order to have a whole scenario on the methylation of the transgene. Five different PCR fragments have been analysed, fragment 0 amplified from the bottom strand corresponds to the 35S promoter region and four fragments covering almost the complete transcribed region, two fragments (I and II) were amplified from the top strand of the transgene and two other fragments (III and IV) from the bottom strand (Fig. 1C). In total, methylation of 38 clones from two independent non-infected plants were examined and only 1 out of 2183 citosine residues was potentially methylated. These results reflect first that the bisulfite treatment efficiently converted non-methylated cytosine residues to uracil, and second that the transgene DNA in non-silenced non-infected NIbV plants was not methylated. Similarly, methylation of 65 clones of five PCR fragments from three independent regenerated recovered

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plants (REG) were analysed, at least two PCR amplifications for each fragment from two independent plants was carried out. The clones derived from fragment 0 corresponding to the 35S promoter region (Fig. 1C) showed no methylation: a total of 608 cytosine residues in 16 clones were examined and only 1 C residue was identified as potentially methylated. These results indicate that the promoter region of the transgene in the REG plants was not methylated, so the transgene silencing associated with the recovery was not due to transcriptional gene silencing (TGS). Methylation of the transgene of the REG plants was only apparent in the 49 clones of fragments from the transcribed region, in which 40.2% of the Cs on average were methylated (Fig. 1D). Several features could be noticed in the methylation distribution of the transcribed region. First, the methylation was not evenly distributed throughout the coding region but increases towards the 3´ end. A scarce methylation in fragment I located at the 5´ end (11.3% of Cs) contrasted with a dense methylation at fragment IV at the 3´ end (81.5% of Cs) of the transcribed region (Fig. 1D). Second, although methylation in both symmetric (CG and CHG) and non-symmetric (CHH) sequence contexts was observed, differences in the frequency of methylation of cytosines located at different sequence contexts could be observed. Methylation of Cs located at CG sites seemed to be more efficient than methylation at CHG and CHH sites since the percentage of CG methylation was higher in all fragments (Fig. 1D). This CG site preference was more apparent in fragments with low level of methylation, 61.5% and 71.8% (fragments I and II) on average at CG sites compared to 2.7% and 18.9% (fragment I and II) at CHG sites and 0.6% and 15.2% (fragments I and II) at CHH sites (Fig. 1D). Due to the design of primer combinations used for the PCR amplification of the bisulfite treated DNA, the complementary strand was also amplified from regions with extensive methylation. The analysis of this strand also showed similar levels of methylated citosines (Supplementary Fig. 1B).

Analysis of transgene expression in NIbV plants after different events of infection, regeneration and sexual reproduction. In order to further examine the relationship between methylation of the transgene coding region and RNA silencing in NIbV plants, we designed a complete experiment to obtain different silenced and non-silenced plants to analyse the methylation status of the transgene and its heritability (Fig. 2). As a control, plants were regenerated in vitro from leaf explants from non-infected NIbV tissue. The analysis of the transgene mRNA accumulation by Northern-blot showed that two types of plants were obtained (Fig. 2 and 3A). Two out of four plants (REGNI plants #25 and #30, pool E) behaved as the parental plants (NI plants #25 and #30), they were non-silenced and showed

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similar levels of transgene mRNA accumulation (Fig. 3A). Nevertheless, the other two plants (REGNI plants #22 and #29, pool D) differed from the parental plants (NI plants #22 and #29, pool A) in which they were silenced since low accumulation of transgene mRNA was observed by Northern blot analysis (Fig. 3A). Bisulfite sequencing analyses of these two types of plants (pool D and E) showed differences in methylation between active and silenced transgene in fragment VI at the 3´end region. A scarce methylation in non-silenced plants of pool E (3.5 % of Cs) contrasted with a dense methylation in silenced plants of pool D (86 % of Cs) (Supplementary Fig. 2) similar to that found in REG plants (Fig. 1D). These results indicated that transgene silencing had been induced most likely during the tissue culture procedure used to obtain the regenerated plants although not in all the plants possibly due to the complex transgene configuration of the transgenic line. It has been described that cell culture might influence global methylation patterns including transgenes (Fojtova et al. 2003; Kaeppler et al. 2000; Koukalova et al. 2005; Meng et al. 2006). Interestingly, the silenced status of the REGNI plants #22 and #29 was not inherited since their progeny showed similar levels of transgene mRNA accumulation than the former parental plants (Fig. 3A) suggesting that the RNA silencing induced in these plants was reset after meiosis as it was described for plants regenerated from recovered tissue from PPV-infected NIbV plants (Guo et al. 1999).

Further samples to analyse transgene silencing and the DNA methylation status of the transgene were collected as follows, sample B of non-symptomatic tissue was collected from infected plants after recovery (Fig. 2). Two sets of regenerated recovered plants (REG) (samples C and F) were obtained after in vitro regeneration from recovered tissue of three independent PPV-infected NIbV plants (#1, 3 and 15) (Fig. 2). Sample C was obtained after one round of in vitro regeneration and sample F was obtained after a subsequent in vitro regeneration from plants of sample C and further vegetative multiplication from stem cuts for up to 11 months (Fig. 2). Successive progenies of the above samples were also collected in pools (4 plants each), samples M, N and O corresponded to the 1st progeny of the 2nd REG plants and samples P, R, S and T corresponded to their 3rd progeny. Progenies of the REG plants were collected as well (1st progeny REG) (Fig. 2). As expected, the analysis of the transgene mRNA accumulation by Northern-blot showed that both in the recovered tissue (sample B) and in the REG plants (samples C and F), low accumulation of the mRNA was detected, indicating that virus induced gene silencing had taken place in the recovered tissue and maintained after the regeneration processes (Fig. 3B). It was also checked that sibling plants from samples C and F showing post-transcriptional gene silencing were also resistant to PPV infection, indicating that the induced silencing was also accompanied of the corresponding resistance phenotype. Furthermore, the silenced status of

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plants from samples B and F was not meiotically stable since their progeny accumulated mRNA of the transgene to the levels of the former parental plants (Fig. 3B) and behave as non-infected NIbV plants in which they were susceptible to PPV infection and recovered from infection as well. No major differences in the level of transgene mRNA accumulation were observed in the sexual descendants of infected/recovered plants (1st progeny REC) and in the 1st (M, N and O) and 3rd (P, R, S and T) progenies of the 2nd REG plants (Fig. 3C).

Analysis of transgene DNA methylation in silenced and non-silenced tissues. Next, we analysed the DNA methylation of the transgene in the plants depicted in Fig. 2. Based on the methylation pattern found in the first methylation analyses (Fig. 1), a fragment at the 5´ end of the transcribed region, with low level of methylation (fragment V), and at the 3´ end, with high level of methylation (fragment VI), were chosen to be analysed (Fig. 4A). As expected, in non-infected plants (pool A) only 5 out of 1132 Cs analysed from both regions were methylated consistent with previous data showing that non-silenced tissue had nonmethylated transgene DNA. Interestingly this methylation did not seem to be a stochastic background since the five Cs methylated were in the symmetrical CG context. This low level of methylation in CG context has been previously described in non-silenced transgenic lines (Haque et al. 2007). The general pattern of methylation found in both regions in the silenced tissue of recovered plants (pool B), 1st and 2nd REG plants (pools C and F, respectively) was very similar to the pattern found in the REG plants from the previous experiment (Fig. 1D and 4B). Fragment V had a overall lower level of methylation in all the silenced samples, 4.6% in pool B, 15.5% in pool C and 13.9% in pool F when compared to fragment VI, where most of the Cs were methylated, 79.4% (pool B), 82.9% (pool C) and 75% (pool F) (Fig. 4B and C). As explained above, due to the design of the primer combinations used for the PCR amplification of the bisulfite treated DNA and the high level of methylation of fragment VI, the complementary strand was also amplified and also showed an extensive methylation (Supplementary Fig. 1C). In general, it was observed that the methylation pattern established in REC plants (pool B) was remarkably stable in the REG plants obtained after a second round of regeneration and up to 11 months of further vegetative multiplication (pool F). Nevertheless slight differences between samples could be observed in these analyses. The level of methylation of fragment V in REG plants (15.5% and 13.9% in pools C and F respectively) is higher than in REC plants (4.6% in pool B) (Fig. 4B and C). Moreover the methylation in samples C and F extended towards the 5´ end of fragment V, which was poorly methylated in sample B (Fig. 4B). In this regard, it is particularly noticeable that the largest increase of methylation occurred at CG sites, from 6.8% (pool B) to 39.4% and 34.5% in (pools C and F respectively) (Fig. 4B and C). It is worth mentioning that methylation of fragment V remained

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low throughout the time (two regenerations plus 11 months of in vitro propagation) without reaching the high levels of methylation observed in fragment VI.

Analyses of siRNA accumulation in silenced and non-silenced tissues. It is well known that siRNAs is a hallmark of the silencing status (Hamilton et al. 2002), and in many cases DNA methylation also depends on the presence of RNA signals consisting by small RNA molecules (Matzke et al. 2007). To gain further insight in the process of silencing and methylation, the accumulation of siRNAs in silenced and non-silenced tissues have been analysed by deep-sequencing analysis. In total 5514879 trimmed sequences have been obtained from non-infected NIbV tissue (NI) and 5437067 from REG plants (Supplementary Table 1). When considering total reads, we observed in both samples a major peak at 24nt, and a second peak at 21nt (Supplementary Fig. 3A) as previously reported for wild type N. benthamiana plants (Valli et al. 2011). In contrast, when only sequences matching the transgene NIbV were considered, 21- and 22-nt species were the major peaks observed (Supplementary Fig. 3B). In order to compare siRNA profiles from both samples, the reads matching the transgene were normalized to the total number of reads for each data set. The number of reads in the 35S promoter region of the NIbV transgene was very low and did not increase after infection and recovery (Supplementary Table 1). We observed an increase in the number of reads in the NIb region in REG plants, with a FoldChange of 4.1 on average compared with non-silenced tissue (NI), (Fig. 5). Although the largest difference in accumulation, 5.3-fold increase, was obtained with the methylation associated 24nt species, it is not significantly different when compared to 21- and 22- species (4-fold and 3.4-fold respectively). Importantly, although silencing caused an increase in the amount of siRNAs both at the fragment V and VI regions, the effect was more apparent in the upper region, which shows lower level of methylation after transgene silencing (Fig. 4, 5B and Supplementary Table 2). These results indicate that whereas both siRNA accumulation and DNA methylation have increased in REG plants, local enhancement of methylation is not associated with a specific enrichment in siRNAs accumulation.

Analysis of transgene DNA methylation inheritance. As shown above, the progenies of the regenerated recovered plants have lost the silenced status of the transgene and the levels of transgene mRNA accumulation were comparable to those of non-infected plants (Fig. 3C), as a consequence the progenies were susceptible to PPV infection. In order to check whether the transgene DNA methylation was also lost, we analysed the methylation of the sexual progenies of REG plants derived from two steps of in vitro

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regeneration. Three pools of 4 plants (M, N and O) from the 1st progeny and two pools (P and S) from the 3rd progeny were subjected to bisulfite sequencing (Fig. 2 and 6). Fragment V and VI corresponding to the 5´ and 3’ ends of the transcribed region of the NIbV transgene were analysed (Fig. 6A). Although all progenies of the regenerated recovered plants were not silenced as the non-infected plants, the bisulfite sequencing showed that, unlike the non-infected tissue (Fig. 4B, pool A), 1st and 3rd progenies had a certain level of methylation (Fig 6B). In fragment V, 13.8% of Cs in the 1st progeny and 11.4% in the 3rd progeny were methylated, and in fragment VI 6.9% and 14.9% in the 1st and 3rd progeny, respectively (Fig. 6B and C). The methylation pattern observed in the 1st and the 3rd progeny were comparable, but they were very different from the pattern of the silenced tissue established following VIGS and in vitro regeneration (Fig. 4B and 6B, pool F). The asymmetric CHH methylation was almost completely lost along the transgene after meiosis even in regions with dense methylation. With respect to symmetric methylation at CHG sites, in the 3´ end region it was drastically reduced after the first sexual propagation to low levels similar to those of the 5´ end of the coding region. But after three generations, the CHG methylation only remained in the 3´ end of the transgene transcribed region. Interestingly, in contrast with the almost absence of methylation of the transgene in non-silenced non-infected plants (Fig. 4B, pool A), methylation in CG sites was conserved high at both ends of the transcribed regions of the transgene in the progenies of regenerated recovered plants (on average 59% in 1st progeny and 81.2% in 3rd progeny, Fig. 6C). Methylation in CG sites at the 5’ end of the transcribed region was even higher in the nonsilenced sexual offsprings than in the silenced recovered plants from which they descend (Fig. 2 and 6B). These results indicate that the silencing-associated methylation pattern established after VIGS was not inherited after sexual propagation but changed to a CG methylation pattern which was not associated to silencing but was inherited instead.

DISCUSSION DNA cytosine methylation is thought to act primarily as a defense system against the transcriptional activity and mobility of transposable elements (Kato et al. 2003; Miura et al. 2001). Likewise, DNA methylation and RNA silencing in TGS and PTGS would operate as a defense mechanism against invading nucleic acids such as viruses and transgenes (Plasterk 2002; Voinnet 2001). More recently, advances in microarray and high-throughput sequencing have revealed the unexpected wide presence of methylation in the transcribed regions of endogenous genes. There is no question to affirm that genic transcription and DNA methylation are closely interwoven processes (Bender 2004; Furner and Matzke 2011; Lister et al. 2008), but so far the statement that DNA methylation usually silences gene expression is only fully true when it takes place in promoters and genes are therefore inactivated by TGS (Mette et al.

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1999; Vaucheret and Fagard 2001). On the contrary, the significance and effects of methylation within the gene body in transgenes silenced by PTGS or in active endogenous genes (Dalakouras et al. 2012; Teixeira and Colot 2009) have not been as thoroughly studied and we are still far from understanding how and when they are activated and its role in gene regulation. The aim of this work was to examine the transgene silencing and DNA methylation that take place in the transgenic line NIbV after VIGS, a particular case of sense PTGS, characterization of the siRNA population associated to the changes and finally to assess the inheritance of these changes. This system is of particular interest because it allowed studying in the same transgenic line the changes associated with transgene silencing by comparing transgene expression and methylation before and after the silencing trigger, PPV infection. Data about patterns of methylation associated with PTGS or VIGS are scarce and only few of them have been analysed in detail using sensitive methods such as bisulfite sequencing (Dalakouras et al. 2012; Haque et al. 2007; Lunerová-Bedřichová et al. 2008; Wang et al. 2008; Wang et al. 2001). Our data showed that PPV-induced silencing of the NIbV transgene caused specific DNA methylation. It was excluded from the promoter region, and weakly affected the 5’ region of the coding sequence where it shows a clear preference for cytosines in CG sites. In the 3’ half of the coding region the methylation in CHG and CHH contexts increased gradually towards the 3´ end until reaching a dense methylation. The observation of a heavy methylation at 3´ end of the coding region is in agreement with previous data on cis-methylation of constitutively silenced sense transgenes (Haque et al. 2007) and on trans-methylation on a transgene that was post-transcriptionally silenced by an invertedly repeated silencer locus (Lunerová-Bedřichová et al. 2008). It has been shown that transgene methylation initiated at the 3’ end can spread upstream, reaching the promoter region and causing transcriptional silencing (Melquist and Bender 2003). This feature has not been observed in the regenerated recovered plants. Although the methylation of the 5’ region of the coding sequence of the transgene was enhanced after regeneration of plants from the recovered tissue of infected NIbV plants, particularly in the CG context (Fig. 4), it did not spread into the promoter region after a second regeneration step and prolonged vegetative propagation in vitro, suggesting that epigenetic switch from PTGS to TGS depends on specific features of the transgene. High-throughput methylome analyses have revealed extensive CG methylation in the coding sequence of a large number of plant genes transcriptionally active (Cokus et al. 2008; Lister et al. 2008). In contrast, the dense methylation of cytosines in CHG and CHH context appears to be a hallmark of RNA-directed DNA methylation resulting in gene silencing (Lunerová-Bedřichová et al. 2008; Pelissier et al. 1999). However, virus-induced extensive methylation in all sequence contexts of a transgene coding for a replicating satellite RNA was associated with only a low reduction in transgene mRNA levels (Wang et al. 2001). This feature

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has also been observed in an endogenous gene, the phosphoribosylanthranilate isomerase (PAI) 1 gene, which is expressed despite showing dense methylation (Melquist and Bender 2003). On the other hand, although DNA methylation was associated with RNA silencing of a Sweet potato feathery mottle virus transgene, grafting of the silenced transgene can induce RNA silencing in trans without inducing methylation (Haque et al. 2007). Thus, although induction of cytosine methylation in non-CG sites and RNA silencing appear to be closely related, RdDM can be separated from efficient RNA silencing in some particular cases.

Which are the elicitors directing RdDM upon RNA silencing induction? Whereas double stranded small RNAs, mainly of 21 and 22nt have been proven to be the molecules involved in specific RNA degradation, in spite of extensive research, the effective inducers of RdDM have not been defined yet (Dalakouras and Wassenegger 2013). There is abundant information showing a very relevant role of 24-nt siRNAs in RdDM (Chan et al. 2004; Melnyk et al. 2011; Molnar et al. 2010; Xie et al. 2004), however, the identification of these molecules as the direct elicitors of DNA methylation is not fully supported by some experimental evidence (Dalakouras and Wassenegger 2013, Melnyk et al. 2011; Mlotshwa et al. 2010). Although deep sequencing analysis cannot be considered an absolute quantitative technique, a large amount of small RNAs (0.36% of small RNAs) mapping in the viral transgene reveals a hot spot in non-infected NIbV plants despite the transgene was active and non-methylated, and the plants are susceptible to PPV infection. The presence of small RNAs that could direct RISC to degrade the transgene mRNA, suggests that the NIbV transgene may be indeed partially silenced, but below the threshold that turns off effectively the transgene expression and provides antiviral resistance. The amount of NIbV-specific small RNAs raised more than four times (1.49% of the total) after PPV infection and recovery, this could reflect the enhancement of the RNA silencing response above the threshold required to maintain the transgene inactivated and methylated and to confer virus resistance to the plant. In contrast with the predominance of 24-nt molecules among endogenous N. benthamiana siRNAs, transgene siRNAs were mainly of 21 and 22nt, and no significant enrichment in 24-nt species was observed after PPV infection and recovery. Moreover, we found no correlation between 24-nt siRNAs and dense transgene methylation at CHG and CHH sites in the recovered NIbV transgenic plants. This is in agreement with the possibility that RdDM induced by PPV infection could have been initiated by a recently described pathway that depends on RDR6 and 21-22-nt siRNAs (Nuthikattu et al. 2013; Pontier et al. 2012), which is also expected to occur in the sense post-transcriptional silencing of the NIbV transgene. Since similar amounts of 21-22-nt siRNAs matched fragments V and VI in NIbV recovered plants, the prevalent methylation of fragment VI could be consequence of specific recognition of RDR6 of the 3’end of the transgene mRNA. Since the minor 24-nt

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species also increased by the PPV-induced silencing, it is likely that the classical Poll IVmediated pathway of RdDM could contribute to maintain the methylation of the NIbV transgene, as it has been suggested for loci whose methylation was initiated by the RDR6mediated pathway (Nuthikattu et al. 2013). Nuclear viroids and cytoplasmic RNA viruses, including PPV, have the ability to direct de novo DNA methylation associated with RNA silencing (Guo et al. 1999; Jones et al. 1998b, Wassenegger et al. 1994). Plants regenerated from recovered tissue of PPV-infected NIbV plants did not show PPV siRNAs mapping outside the NIbV transgene above the very low background found in healthy plants. However they maintained specific virus resistance, transgene silencing and dense transgene methylation. This reflects that the signal for maintenance of this specific phenotype is produced in the regenerated plants and it was independent of the trigger viral replication.

In contrast with the maintenance of dense transgene methylation after vegetative propagation of NIbV plants in the absence of PPV replication, the asymmetric methylation of the NIbV transgene was lost after sexual propagation of the recovered plants. This is in agreement with previous reports on virus-induced methylation (Guo et al. 1999; Jones et al. 1998b) and with a recent report showing that viroid-induced asymmetric methylation was lost upon losing the viroid trigger by transgene segregation (Dalakouras et al. 2012). Thus, upon losing the initial trigger RdDM is not maintained in the sexual progeny as a consequence of genome-wide losses of DNA methylation during male and female gametogenesis (Law and Jacobsen 2010). Virus-induced transgene silencing is also lost during sexual reproduction, supporting that signals induced by the same trigger to maintain RNA silencing and RdDM are functionally related. Until recently the generally accepted idea was that only methylation associated to TGS was sexually inherited (Jones et al. 2001). The use of the very sensitive technique of bisulfite sequencing has shown that, whereas dense methylation in CHH is lost, CG methylation, an to lower extent CHG methylation, can be inherited by the progeny of plants that have undergone IR- (Lunerová-Bedřichová et al. 2008) viroid- (Dalakouras et al. 2012) or virus- (this work) induced gene silencing. The inherited CG methylation pattern is not associated with transgene silencing or virus resistance. In agreement with genome-wide methylome studies showing that methylation at CG sites of coding regions is abundant in expressed endogenous genes (Teixeira and Colot 2009). Although current information does not allow to conclude whether dense methylation including citosines in asymmetrical context contribute to induce or maintain RNA

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silencing, it appears clear that CG methylation, whose maintenance does not depend on RdDM signals, has not a major role in silencing endogenous genes or transgenes. The choice of genome sequences to be methylated has received much attention over the last two decades. Despite its wide presence, neither the biological function of gene body methylation nor the molecular bases by which genes become methylated are understood. As suggested by Lunerová-Bedřichová et al. (2008), it is tempting to speculate that a CG methylation pattern could be considered a hallmark of an ancient event of PTGS or VIGS, associated to RdDM at both symmetrical and non-symmetrical sites. It remains to be shown if the remaining CG methylation could confer any adaptative advantage to the progeny which receive this type of epigenetic information. Moreover, understanding methylation patterning in body genes and its influence in silencing will help to know whether a gene with a certain methylation pattern would be silenced or had undergone silencing in the past and, most interestingly, will help to engineer silencing à la carte for achieving plants with altered traits.

EXPERIMENTAL PROCEDURES Virus and plants The PPV isolate (PPV-R) and the transgenic N. benthamiana line NIbV-3, transformed with a fragment of the PPV-R genome and inserted in two loci have been described previously (Guo and García 1997). Transgenic plants were grown from seeds germinated in kanamycin (0.1 mg/ml). After leaf disks sterilization in 10% commercial bleach (hypoclhorite) for 5-10 min and rinsing with sterile H2O, plants were regenerated by in vitro culture on solid Murashige and Skoog (MS) medium containing 6-benzylaminopurine (1mg/liter), α-naphthalen acetic acid (0.1 mg/liter) and kanamycin (0.1 mg/ml) and rooted on MS medium.

Bisulfite genomic sequencing Genomic DNA was purified with Genomic-tips100/G according to manufacturer´s instructions (Qiagen). The first four fully developed leaves were collected from non-infected plants and plants of the progenies. Non-symptomatic upper leaves from recovered plants were collected after 28 days post-inoculation. Leaves from explants 10-15 0cm long from REG plants were collected. Prior to treatment with bisulfite, two micrograms of SalI digested genomic DNA, phenol/chloroform extracted, ethanol precipitated and water resuspended. Bisulfite treatment was performed according to the method described by (Clark et al. 1994). Briefly, the digested DNA was first heat-denatured at 100ºC, 5 min, cooled for 5 min, and denatured in 0.3

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M NaOH for 15 min at 75ºC in 20 µl volume. Denatured DNA was mixed with 200 µl of a freshly prepared 2 M sodium metabisulfite (Merck)/50mM hydroquinone (Sigma), pH5.0 (1.7M/0.5mM, final concentrations). The reaction mixture was incubated in a thermal cycler (MJ Research) for 6 h at 50ºC with a denaturation step at 94ºC every hour. DNA was desalted with a column (Qiagen) and then incubated in 0.3 M NaOH for 20 min at 37ºC. After neutralization with ammonium acetate to a final concentration of 3 M, DNA was ethanol precipitated and resuspended in 70 µl of water.

The bisulfite-treated DNA (5µl) was amplified by PCR in 20µl containing 40 pmol each primer, 0.1 mM each dNTP and 2.5 units of Taq (Perkin-Elmer) in the recommended buffer. The PCR program consisted of 30 cycles of 92ºC for 30 sec, annealing at the Tmh (the highest Tm of the primers) for 30 sec; temperature decreased 2ºC/each cycle until reach the Tml (the lowest Tm of the primers), 72ºC for 1 min and finally 4ºC. PCR primers were designed to allow amplification of methylated, unmethylated and partially methylated targets for the upper and lower strands. The primers contained degenerate nucleotides, either cytosine or thymine (Y) for forward primers or adenine or guanine (R) for reverse primers (Supplementary Table 3). PCR products were excised from gel and cloned in TOPO-TA vector (Invitrogen). At least 6 individual clones were sequenced for each PCR fragment.

RNA isolation and Northern blot analysis Total RNA from leaves was isolated using 1 ml Trizol Reagent (Invitrogen) according to the manufacturer´s instructions. Northern-blot analysis was performed as described (Simón-Mateo et al. 2003).

Deep sequencing analysis of small RNAs Total RNA from leaves was isolated using 1ml Trizol Reagent (Invitrogen) according to the manufacturer´s instructions. The production of small RNA libraries was done according Valli et al. (2011). Nucleotide sequence of the amplified cDNA was analysed using Illumina Analyzer. The resulting short reads were filtered to discard tRNAs and rRNAs and mapped to the transgene region allowing only perfect matches.

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ACKNOWLEDGEMENTS We thank Elvira Domínguez and Beatriz García for technical support and David Baulcombe for his helpful collaboration in the smallRNA sequencing. This work was supported by grants BIO2010-18541 (Spanish MEC) and KBBE-204429 (EU). The authors have no conflict of interest to declare.

LEGENDS LEGENDS FOR SUPPORTING INFORMATION Supplementary Figure 1. Distribution of cytosine methylation of the transgene complementary strand of recovered tissue and regenerated silenced plants. Supplementary Figure 2. Distribution of cytosine methylation of the transgene of regenerated silenced (pool D) and non-silenced (pool E) plants obtained from non-infected NIbV plants. Supplementary Figure 3. Size distribution of small RNA populations. Supplementary Table 1. Number of small RNAs mapped to different regions of the transgene unit allowing only perfect matches. Supplementary Table 2. Small RNAs mapped to different regions of the transgene unit allowing only perfect matches. Supplementary Table 3. Primers used for PCR amplification.

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Figure 1. Distribution of cytosine methylation of the transgene of plants regenerated from recovered tissue of a PPV-infected NIbV plant (REG). A) Schematic representation of the experimental procedure to regenerate silenced plants. B) Transgene mRNA accumulation in pools of three plants: non-infected plants (NI), recovered tissue (REC) and regenerated recovered plants (REG). Methylene blue staining of the membrane was used to check loading (control). C) Schematic representation of the transgene unit in which fragments analyzed by bisulfite sequencing is indicated. Fragments amplified from the bottom strand are in dark and from the top strand in light. 35S: promoter; NCR: PPV 5´ non-coding region; NIbV: NIb coding region. D) The graphs show the percentage of methylation at individual cytosines of fragments 0, I, II, III and IV in REG plants. Distribution of the cytosines in symmetrical (CG, CHG) and non-symmetrical (CHH) contexts is shown below the graphs. Histograms representing the percentage of methylation are shown in the lower part of the figure. Figure 2. A) Schematic representation of the experimental procedure to obtain plants from nonsilenced NIbV plants with different silencing status of the transgene. NI: non-infected NIbV plants, REC: recovered tissue from a PPV-infected plant, 1st REG: plants regenerated from recovered tissue, 2nd REG: plants regenerated from the first regenerated plants, progeny: progeny from the indicated plants. The plants analyzed in pools A, B, C, D, E and F is indicated. Four plants of each progeny were analyzed in pools M, N, O, P, R, S and T. The expression of the transgene in the plants (silenced or non-silenced) is indicated. Figure 3. Transgene mRNA accumulation of plants described in Figure 2. A) Non-infected (NI) NIbV plants #22 and 29 (pool A), 25 and 30; plants regenerated from non-infected tissue [plants #22 29 (poolD), and 25 and 30 (pool E)] (REGNI) and progeny (prog) from regenerated plants #22 and 29. NT: non-transgenic plant. B) Recovered tissue (REC) from PPV-infected NIbV plants #1, 3 and 15 (pool B) and their progenies (1st prog REC, plants #1, 3 and 15).

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Regenerated recovered plants #1, 3 and 15 (1st REG, pool C) obtained from recovered tissue. Regenerated recovered plants #1, 3 and 15 (2nd REG, pool F) obtained from 1st REG plants #1, 3 and 15, and their progenies in pools (four plants each) M, N and O (1st prog 2nd REG). C) Progenies from PPV-infected NIbV plants #1, 3 and 15 (1st prog REC). First (M, N and O plants) and 3rd (pools P, R, S and T, four plants each) progenies from plants obtained by two regeneration steps from recovered tissue (2nd REG). NT: non-transgenic plant. Methylene blue staining of the membrane was used to check loading (control). Figure 4. Distribution of cytosine methylation of the NIbV transgene in non-infected plants, tissue recovered after infection and regenerated silenced plants. A) Schematic representation of the transgene unit in which fragments analyzed by bisulfite sequencing are indicated. Fragments in blue indicate they are amplified from the top strand. 35S: promoter; NCR: PPV 5´ non-coding region; NIbV: NIb coding region. B) The graphs show the percentage of methylation at individual cytosines of fragments V and VI of the transgene in plants described in Figure 2. NI Pool A: Non-infected NIbV plants; REC Pool B: asymptomatic tissue from recovered PPV-infected NIbV plants; 1st REG Pool C: plants regenerated from recovered tissue; 2nd REG Pool F: plants regenerated from the first regenerated plants. Distribution of the cytosines in symmetrical (CG, CHG) and non-symmetrical (CHH) contexts in each fragment is shown. C) Histograms representing the percentage of methylation from pools B, C and F. Figure 5. Abundance of small RNAs (total, 21nt, 22nt and 24nt) from non-infected NIbV (pool A in Figure 2) (in light) and plants obtained by two regeneration steps from recovered tissue (2nd regeneration, pool F in Figure 2) (in dark). A) Histograms of sequences mapped to transgene unit allowing only perfect matches. B) Histograms of sequences mapped to Fragment V and VI allowing only perfect matches. The fold increase of small RNA species in regenerated recovered plants (in dark) compared to non-infected NIbV plants (in light) is shown above each graph. Figure 6. Distribution of cytosine methylation of the NIbV transgene in regenerated silenced plants and their progenies. A) Schematic representation of the transgene unit in which fragments analyzed by bisulfite sequencing are indicated. Fragments in light indicate they are amplified from the top strand. 35S: promoter; NCR: PPV 5´ non-coding region; NIbV: NIb coding region. B) The graphs show the percentage of methylation at individual cytosines of fragments V and VI of the transgene in different plants described in Figure 2. 2nd REG Pool F: plants obtained by two regeneration steps from recovered tissue; 1st progeny pools (four plants each) M, N, O: 1st progeny from the 2nd REG plants; 3rd progeny pools (four plants each) P, S: 3rd progeny from the 2nd REG plants. Distribution of the cytosines in symmetrical (CG, CHG) and non-symmetrical (CHH) contexts in each fragment is shown. C) Histograms representing the percentage of methylation from pools F, M, N, O, P and S.

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