Changes in the DNA methylation pattern of the host male gametophyte of viroid-infected cucumber plants
Journal of Experimental Botany
Changes in the DNA methylation pattern of the host male gametophyte of viroid-infected cucumber plants
Mayte Castellano 1
German Martinez 0
Maria Carmen Marques 1
Jordi Moreno-Romero 0
Claudia Köhler 0
Vicente Pallas 1
Gustavo Gomez 1
Ramanjulu Sunkar, Oklahoma State University
0 Department of Plant Biology, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology , SE-750 07 Uppsala , Sweden
1 Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Politecnica de Valencia (UPV) , CPI, Edificio 8 E, Av. de los Naranjos s/n, 46022 Valencia , Spain
Eukaryotic organisms exposed to adverse conditions are required to show a certain degree of transcriptional plasticity in order to cope successfully with stress. Epigenetic regulation of the genome is a key regulatory mechanism allowing dynamic changes of the transcriptional status of the plant in response to stress. The Hop stunt viroid (HSVd) induces the demethylation of ribosomal RNA (rRNA) in cucumber (Cucumis sativus) leaves, leading to increasing transcription rates of rRNA. In addition to the clear alterations observed in vegetative tissues, HSVd infection is also associated with drastic changes in gametophyte development. To examine the basis of viroid-induced alterations in reproductive tissues, we analysed the cellular and molecular consequences of HSVd infection in the male gametophyte of cucumber plants. Our results indicate that in the pollen grain, accumulation of HSVd RNA induces a decondensation of the generative nucleus that correlates with a dynamic demethylation of repetitive regions in the cucumber genome that include rRNA genes and transposable elements (TEs). We therefore propose that HSVd infection impairs the epigenetic control of rRNA genes and TEs in gametic cells of cucumber, a phenomenon thus far unknown to occur in this reproductive tissue as a consequence of pathogen infection.
Cucumber; epigenetic inheritance; hop stunt viroid; viroid-plant interactions; viroid-induced pathogenesis; viroids and DNA methylation
The maintenance of genome stability is a constant
requirement in living organisms. At the same time, however,
organisms must ensure that a certain level of genome plasticity is
available in order to allow for genome rearrangements and
mutations that might introduce beneficial traits to cope with
stresses. In the case of plants, their sessile nature means
that they are constantly exposed to different biotic and
abiotic stresses that very often affect genome stability in both
somatic and meiotic cells (Boyko and Kovalchuk, 2011; Zhu
et al., 2016). Viroids are pathogenic long non-coding RNAs
(lncRNAs), able to infect and systemically invade
herbaceous and ligneous plants (Flores et al., 2005; Ding, 2009;
Gomez and Pallas, 2013). Constrained by their small
(250–400 nts) and non-protein-coding genome, viroids have
© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
evolved into versatile nucleic acids that subvert the plant-cell
machinery at diverse functional levels in order to guarantee
that their life cycle can be completed within the infected host
(Ding, 2009). Although some pathogen–host interactions
occur without visible plant alterations (latent diseases), viroid
infection is frequently associated with phenotypic changes
that we recognize as symptoms.
Because these pathogenic RNAs lack protein-coding
activity, it was initially assumed that viroid-induced symptoms
resulted from a direct interaction between specific structural
elements of the viroid RNA genome and certain host factors
(proteins or nucleic acids) (Ding, 2009; Navarro et al., 2012).
However, in recent years an increasing amount of
experimental data has provided evidence for the existence of other
potential pathogenic mechanisms, for example the close
interplay between viroid-induced pathogenesis and RNA silencing.
The initially proposed idea that certain viroid-derived small
RNAs (vd-sRNAs) can down-regulate in trans host mRNAs
to promote expression of symptoms (Papaefthimiou et al.,
2001; Wang et al., 2004; Gomez et al., 2009) was
experimentally validated for members of both Pospiviroidae (Gomez
et al., 2008; Eamens et al., 2014; Adkar-Purushothama
et al., 2015) and Avsunviroidae (Navarro et al., 2012) families.
The observation that the Cucumber mosaic virus Y-satellite
RNA uses a similar mechanism to alter host-gene expression
(Shimura et al., 2011; Smith et al., 2011) suggests that this
pathogenesis strategy is not exclusive for viroids. At a
different functional level, it is also recognized that in addition to
viroids, viruses, bacteria, nematodes, and aphids can alter the
miRNA (Ruiz-Ferrer and Voinnet, 2009; Garcia and Pallas,
2015) or sRNA-metabolism (Cao et al., 2014) of their host
Recent studies have produced evidence of global changes
of the epigenetic regulation of the host-genome upon viroid
infection. An examination of the interaction of Hop stunt
viroid (HSVd) with two different hosts (Cucumis sativus,
cucumber, and Nicotiana benthamiana) showed that
viroidaccumulating plants exhibit an increased rRNA transcription
rate. This altered transcription was associated with reduced
cytosine methylation of rDNA promoter regions, revealing
that some (normally silenced) rRNA genes are
transcriptionally reactivated during HSVd infection (Martinez et al., 2014;
Castellano et al., 2015). However, induction of changes in the
host epigenome is not exclusive for viroid infection. Indeed,
dynamic changes in host-DNA methylation patterns occur
during antibacterial or antiviral defence in rice (Sha et al.,
2005), tobacco (Boyko et al., 2007), and Arabidopsis (Dowen
et al., 2012; Yu et al., 2013). Furthermore, overexpression
of the replication-associated protein (Rep) of a geminivirus
has been shown to induce hypomethylation of host DNA
in N. benthamiana plants (Rodriguez-Negrete et al., 2013).
Taken together, these observations support the notion that
host-DNA demethylation may be part of a common induced
immune response in plants (Alvarez et al., 2010; Zhu et al.,
In viroid–cucumber interactions DNA demethylation has
been connected with HSVd recruiting and functionally
subverting the host HISTONE DEACETYLASE 6 (HDA6)
(Castellano et al., 2016). In Arabidopsis HDA6 confers an
epigenetic memory of the silent state (Blevins et al., 2014) and is
furthermore involved in the maintenance and de novo CG and
CHG (where H is A, T or C) methylation of transposable
elements (TEs), rRNA genes, and transgenes via its interaction
with DNA METHYLTRANSFERASE 1 (MET 1) and the
RNA-directed DNA methylation (RdDM) pathway (Aufsatz
et al., 2002; Probst et al., 2004; Earley et al., 2010; Liu et al.,
2012; Hristova et al., 2015). Viroids are pathogenic, long
noncoding RNAs (lncRNAs) that subvert endogenous
lncRNAdirected regulatory routes to complete their life cycle in the
infected cell (Gomez and Pallas, 2013). Remarkably,
endogenous lncRNAs are able to function as epigenetic modulators
by binding to chromatin-modifying proteins and recruiting
their catalytic activity to specific sites in the genome, thereby
modulating chromatin states and impacting on gene
expression (Mercer and Mattick, 2013).
HSVd is a polyphagous pathogenic lncRNA that is able to
infect a wide range of hosts (including cucumber, grapevine,
citrus, plum, and peach) and causes diverse symptoms (Pallas
et al., 2003; Sano, 2003). In cucumber, as well as causing plant
stunting, HSVd infection induces severe alterations in
reproductive organs that are frequently associated with reduced
fertility (Singh et al., 2003; Martinez et al., 2008). Although
HSVd is poorly transmitted through seeds and pollen, seed
transmission may play a role for its survival in certain hosts
such as grapevine (Wah and Symons, 1999). The molecular
mechanisms underlying the structural and functional
alterations in host reproductive organs associated with viroid
infection are currently unknown.
Having established that HSVd alters DNA methylation in
vegetative cells, in this study we addressed the question as to
whether the epigenetic changes induced by viroid infection
are also present in the male gametophyte. Pollen grains are
known to transmit other members of the Pospiviroidae
family (Singh, 1970; Kryczyński et al., 1988; Barba et al., 2007;
Card et al., 2007). Our results reveal that both HSVd mature
forms and vd-sRNAs can be recovered from pollen grains
of infected cucumber plants. Moreover, viroid accumulation
is associated with increased pollen germination levels and
heterochromatin decondensation in the generative nucleus.
Analysis of DNA methylation in rDNA and TE repeats
reveal a significant reduction in the symmetric cytosine
methylation context, which is associated with a transcriptional
increase of their RNAs. In summary, our results show that
previously observed epigenetic changes in vegetative tissues
are maintained in male gametes and are thus passed on to the
Material and methods
Six cucumber (Cucumis sativus cv Marketer) plants were
inoculated with Agrobacterium tumefaciens strain C58C1 transformed
with a binary pMOG800 vector carrying a head-to-tail infectious
dimeric HSVd cDNA (Y09352) (Gomez and Pallas, 2006), as
previously described (Gomez et al., 2008). Three cucumber plants
infiltrated with A. tumefaciens strain C58C1 transformed with a binary
pMOG800 empty vector were used as a mock-inoculated control.
Plants were maintained in growth chambers at 30 °C for 16 h with
fluorescent light and at 25 °C for 8h in darkness until flowering.
Viroid systemic infection in HSVd-inoculated plants was confirmed
by dot-blot hybridization (see Supplementary Fig. S1 at JXB online).
To collect pollen grains, a paintbrush was used to gently brush
pollen from the anthers into an Eppendorf tube. This procedure was
repeated for approximately 750 and 650 mature flowers recovered
from HSVd-infected and control plants respectively, between 80 and
110 d post-infiltration.
As described previously (Aparicio et al., 1999), 20 mg of pollen
grains were suspended in 1.5 ml of phosphate saline-Tween
polyvinylpyrrolidone buffer (pH 7.4), vigorously shaken for 1 min,
and centrifuged at 3000 rpm (1000 g) for 5 min. This procedure
was repeated three times, followed by an additional washing with
1.5 ml of 1% sodium dodecyl sulphate (SDS) to remove particles
firmly bound to the pollen grains. Aliquots from the four
supernatants were phenol extracted and the aqueous phase was ethanol
precipitated and resuspended in sterile water to check for surface
contamination of the viroid. Washed pollen was homogenized for
total RNA extraction. Total RNA was extracted from pooled pollen
grains (~0.1 g) recovered from infected and control cucumber plants
using the TRI reagent (SIGMA, St. Louis, MO, USA) according
to the manufacturer’s instructions. The low-molecular weight RNA
(<200 nt) fraction was enriched from total RNA using MIRACLE
(miRNA isolation Kit, STRATAGENE) according to the
manufacturer’s instructions. Supernatants and washed pollen were analysed
for the presence of HSVd by RT-PCR as described by Martinez
et al. (2010).
Small RNA library information
The sRNA sequences used in this work were obtained from an
sRNAs population recovered from the pollen of mock-inoculated
and Hop stunt viroid-infected cucumber plants. The libraries were
sequenced using a HIseq 2500 system (Illumina Technology).
Bisulfite conversion and sequencing
Total genomic DNA was extracted from pollen grains (~0.1 g)
recovered from different infected and healthy cucumber plants
(Dellaporta et al., 1983). Bisulfite treatment was performed using
the EpiTec Bisulfite kit (Qiagen). The DNA regions to be
analysed and their corresponding oligos were determined using the
MethPrimer software (http://www.urogene.org/methprimer/)
(Li and Dahiya, 2002). Modified DNA was amplified by PCR
using Taq DNA polymerase (Promega). The following primers
were used to amplify by PCR specific regions of rDNA and TE,
respectively: 45s-Fw ATCATAGATTTTTYGAGGGT (position
–80 to –61), 45s-Rv ATGACGACRTAAACATCCCAA
(position +101 to +121) (according sequence X51542.1); and TE-Fw
TAGTTTTTTGAYAGGGGAAATA (position 545 to 566),
TE-Rv CATTCATAAACTTRCTTTCTCA (position 760 to
781) (according TE cucumber predicted sequence: cuc_reannotTE.
Scaffold000159.7) (Li et al., 2011). The amplicons obtained were
cloned using the InsTAclone PCR cloning Kit (Thermo Scientific).
We selected for sequencing sixteen to fifty clones (obtained from two
independent replicates) from rDNA and TE, respectively, for each
analysis in both the HSVd-infected and control pollen.
Total RNA, extracted from pollen grains collected from
HSVdinfected and control plants, was treated with DNase in order to
avoid DNA contamination. Reverse transcription (RT) PCR
analysis of serial dilutions (500, 100, and 20 ng) of total RNAs obtained
from HSVd-infected and control pollen was performed using the
SuperScript® III One-Step RT-PCR System with Platinum® Taq
DNA Polymerase (Invitrogen) according to the manufacturer’s
instructions. The sequence and relative position of the specific
primers used to amplify a region ( ̴160 nt) of rRNA precursor by
RT-PCR are detailed in Supplementary Table 1. RT-PCR
conditions were 45 °C/30 min, 95 °C/15 s, 51 °C/30 s, 72 °C/20 s (30
To amplify the TE transcripts we used the oligos (TE-Dir
TAGCTTTCTGACAGGGGAAATACC, and TE-Rv
GCATTCATGAACTTGCTTTCTCAGC) flanking a region
(~240bp) of an annotated TE cucumber sequence
(cuc_reannotTE.Scaffold000159.7) (Li et al., 2011). RT-PCR conditions
were 45 °C/30 min, 95 °C/15 s, 62 °C/30 s, 72 °C/15 s (30 cycles).
The primers Ub-Dir (5´ CACCAAGCCCAAGAAGATC)
and Ub-Rev (5´ TAAACCTAATCACCACCAGC) flanking a
region (~220 nt) of ubiquitin mRNA (AN: NM-001282241.1)
were used to amplify this mRNA as a load control. RT-PCR
conditions were 45 °C/30 min, 95 °C/15 s, 57 °C/20 s, 72 °C/20 s
(27 cycles). Three repetitions of this analysis were performed.
To discard the possible amplification of residual genomic
rDNA, 100 ng of total RNAs were analysed by PCR using the
primer pairs (Fw-25s CACCAATAGGGAACGTGAGCTG,
Rv-25s GCGCAATGACCAATTGTGCG) flanking a region
(~130 bp) of 25s-rRNA and TE-Dir–TE-Rv, detailed above (see
Supplementary Fig. S2).
Real-time quantitative PCR assays
Total RNAs were extracted from pollen grains as described above.
First-strand cDNA was synthetized by pulsed reverse transcription
(Varkonyi-Gasic et al., 2007) using a RevertAid cDNA Synthesis Kit
(Thermo ScientificTM) as follows. An initial step at 16 °C for 10 min
was followed by 45 cycles of 16 °C for 2 min, 42 °C for 1 min and
50 °C for 1 s, including a final denaturing step at 85 °C for 5min.
qRT-PCR assays were performed using PyroTaq EvaGreen mix Plus
(ROX) (CulteK Molecular Bioline) according to the manufacturer’s
instructions. Ubiquitin mRNA (AN: NM-001282241.1) was used to
Detection of small RNAs was performed starting from low
molecular weight RNA (<200 nt) fractions obtained as described above.
Stem-loop-specific reverse transcription for sRNAs detection was
performed as previously described by Czimmerer et al. (2013) using
a RevertAid cDNA Synthesis Kit (Thermo Scientific). Cucumber
miR159 with stable expression in both analysed samples
according to the sequencing data was used for normalization. All analyses
were performed in triplicates on an ABI 7500Fast-Real Time qPCR
instrument (Applied Biosystems) using a standard protocol. The
efficiency of PCR amplification was derived from a standard curve
generated by four five-fold serial dilution points of cDNA mixed
from the two samples. Gene expression was quantified by the
comparative ΔCt method. The primers used for cDNA synthesis and
qRT-PCR are described above or listed in Supplementary Tables S1
Cucumber reproductive tissues are affected as a
consequence of HSVd-infection
A characteristic symptom related to HSVd infection is an
alteration of fertility that triggers deficiencies in flower size (Sano,
2003; Martinez et al., 2008), fruit quality, and seed viability
(Singh et al., 2003; Martinez et al., 2008). In order to determine
precisely the level of phenotypic effects induced by
HSVdinfection in reproductive organs, we analysed diverse
morphological aspects of cucumber flowers. As shown in the Fig. 1A
flowers (male and female) obtained from HSVd-infected
plants exhibited a significant reduction (close to 50%) of the
corolla-size, as previously observed in the
HSVd–N. benthamiana interaction (Martinez et al., 2008). Morphological studies
showed that HSVd did not induce alterations in pollen grain
size (Fig. 1B). Study of pollen grains by DAPI staining showed
a significant size increase of the generative cell nucleus in
infected cells (Fig. 1C). A more detailed analysis revealed that a
comparable size increase was also observed in the nucleolus of
infected pollen grains (Fig. 1D), suggesting a global alteration
of chromatin structure in pollen grains under viroid infection.
To determine whether this alteration is associated with
physiological changes, we performed germination assays. As shown
in Fig. 1E, pollen grains derived from HSVd-infected plants
had a higher germination rate than pollen collected from
control plants, suggesting that regulatory processes associated with
pollen germination are affected during HSVd-infection.
HSVd accumulates in pollen grains of infected plants
Having established that HSVd induces structural and
functional alterations in the pollen of infected plants,
we attempted to determine if viroid molecules could be
detected in mature dehiscent pollen grains. RT-PCR assays
demonstrated that genomic HSVd RNA accumulated in the
pollen grains obtained from infected plants (Fig. 2A),
indicating that HSVd is able to invade pollen of infected
cucumber plants as has been shown for Potato spindle tuber viroid
(PSTVd) in potato (Singh et al., 1993) and petunia plants
(Matsushita and Tsuda, 2014). We did not detect HSVd on
the surface of the pollen grain (Fig. 2A), providing
robustness to this affirmation.
Given that in cucumber vegetative tissues genomic HSVd
RNA accumulation is associated with the presence of
vdsRNAs (Martinez et al., 2010), we prepared sRNA
libraries from pollen grains derived from mock-inoculated and
HSVd-infected plants. A total of 4 918 251 and 4 566 866
raw sequences (ranging from 18 to 36 nt) were obtained from
HSVd-infected and control pollen grains sRNA libraries,
respectively. Sequences ranging from 20 to 25 nts (2 888 088
for infected pollen and 2 523 747 for the control data set)
were used for further analysis. When sRNAs recovered from
infected and non-infected pollen-grains were analysed by
pairwise alignment against the HSVd genome, we observed
that a total of 18 900 sequences (0.68%; ranging 20 to 25
nts in length) recovered from the infected pollen were
perfectly complementary to HSVd and considered as vd-sRNAs.
Importantly, no sequences perfectly matching with HSVd
were recovered from the control cucumber pollen,
confirming the integrity of the RNA samples. Analysis of polarity
distribution indicated that sRNAs derived from the sense
strand were slightly biased (60%) in comparison to sRNAs
derived from the antisense strand (40%) (Fig. 2B). This
vdsRNA landscape is different to that previously described in
cucumber vegetative tissues where HSVd-derived sRNAs of
both polarities were recovered at comparable levels (Martinez
et al., 2010). Categorized by size, vd-sRNAs were mainly of
22 nt (34.1%), 24 nt (22.1%), and 21 nt (19.9%), whereas
vdsRNAs of 20, 23 and 25 nt amounted to under 6% of the
total vd-sRNAs (Fig. 2B). As previously observed in infected
leaves, sense and antisense vd-sRNAs spreading along the
entire HSVd genome showed a heterogeneous distribution
pattern (Fig. 2C). In summary, pollen grains of HSV-infected
plants accumulate HSVd RNA and vd-sRNAs.
The endogenous sRNA profile is altered in
In vegetative cells of cucumber HSVd infection induces a
drastic change in the accumulation profile of rRNA-derived
sRNAs that is associated with changes in the epigenetic
regulation of those repeats (Martinez et al., 2014; Castellano et al.,
2015, 2016). In order to evaluate whether HSVd infection
induced global alterations in the epigenetic regulation of
pollen, we analysed endogenous sRNAs from pollen of infected
and non-infected plants (Fig. 3A). Viroid-derived sRNAs
recovered from infected pollen were filtered out from this study.
Approximately 85% of the 18–36 nt sRNAs reads (4 219 966
for HSVd-infected and 3 871 276 for mock samples) mapped
with the cucumber genome. These reads were considered as
endogenous pollen sRNAs and used in subsequent analysis. In
HSVd-infected pollen there was a considerable increase of 21,
23, 24, and 25 nt sRNAs (Fig. 3B). In contrast, 22 nt sRNAs
were present at similar frequencies. These results indicate that
HSVd accumulation in pollen grains causes changes in
endogenous sRNAs, resembling – at least in part – those observed in
HSVd-infected cucumber vegetative tissues.
When endogenous sRNAs recovered from infected and
healthy pollen grains were analysed by pairwise alignment
against different Cucumis sativus transcript categories (Li
et al., 2011), we observed that ribosomal and TE-derived
sRNAs were significantly over-accumulated in HSVd-infected
pollen grains (Fig. 3C, Supplementary Figs S3 and S4). This
observation was validated by analysing the accumulation
of representative sRNAs derived from rRNA and TE
transcripts by stem-loop qRT-PCR (see Supplementary Fig. S5).
In contrast, no significant differences were obtained when
sRNAs derived from the coding and centromeric regions
of the cucumber genome were analysed (Fig. 3C). A more
detailed analysis of up-regulated sRNA classes showed that
their over-accumulation in infected samples was detectable in
all size classes (21 to 25 nts) (Fig. 3D). Together, these results
indicate that HSVd accumulation in pollen grains is
associated with alterations in endogenous sRNA levels, mainly
affecting the accumulation of sRNAs derived from repetitive
regions of the genome.
Viroid infection modifies cytosine methylation of
repetitive regions in pollen
To investigate if the increase in sRNAs derived from rDNA
and TEs observed in infected pollen could be linked to
alterations in the host epigenetic landscape, we analysed the
methylation pattern of representative regions of the
repetitive pollen DNAs exhibiting sRNA-derived
over-accumulation. Specifically, we analysed the rRNA promoter region
previously described by Martinez et al. (2014) and a TE
region that had the highest increase in sRNA accumulation.
The genomic DNA extracted from pollen grains of
HSVdinfected and mock-inoculated cucumber plants was
bisulfiteconverted and subjected to PCR to amplify specific regions
of the rDNAs and TE DNA.
We analysed a specific promoter sequence of 201 nt
located between positions –80 and +121 of the 45S-rDNA
containing 16 symmetric (12 CG, 4 CHG) and 23
asymmetric (CHH) potential methylation sites (see Supplementary
Fig. S6A, B). PCR products were cloned, and the sequences
of 51 and 44 clones were compiled for control and infected
samples, respectively. Methylation analysis revealed that
HSVd infection resulted in a significant decrease in the
relative number of total methylated cytosine residues when
compared to the control (Fig. 4A). Hypomethylation was
restricted to symmetric (CG/CHG) sequence contexts
(Fig. 4B, C), and not detected at asymmetric (CHH)
positions (Fig. 4B). We further analysed a 236-bp region of a
TE with homology to a Copia element (termed
cuc_reannotTE.Scaffold000159.7 in the reannotation of TEs from
Li et al., 2011) containing 20 symmetric (9 CG, 11 CHG)
and 41 asymmetric (CHH) potential methylation sites (see
Supplementary Fig. S7A). Similar to the 45S-rDNA locus,
HSVd infection resulted in a significant reduction in the
relative number of methylated cytosine residues of this
transposable DNA compared to the mock-inoculated
controls (Fig. 4D). This drastic hypomethylation affected both
symmetric and asymmetric sequence contexts (Fig. 4E, F)
Hypomethylation of the Copia element in the symmetric
sequence context was also observed in vegetative tissues
of HSVd-infected cucumber plants (see Supplementary
Fig. S6B, C), revealing that this effect was not restricted
to pollen. Taken together, these results indicate that in
pollen grains, similar to vegetative tissues, rDNA and TEs lose
DNA methylation in response to viroid infection,
reinforcing the close interplay suggested to exist between
HSVdpathogenesis and host-epigenetic alterations in these classes
of repetitive DNAs.
HSVd infection promotes transcriptional alterations in
the pollen grain
Silencing of repetitive DNA is a self-reinforcing
transcriptional regulatory phenomenon mediated by
siRNAdirected cytosine methylation and heterochromatin
formation (Slotkin and Martienssen, 2007; Law and
Jacobsen, 2010) that is dynamically regulated during plant
development and stress (Martinez and Slotkin, 2012).
To investigate whether the observed increase of sRNAs
derived from rRNA and TEs and DNA hypomethylation
could be associated with alterations in the host
transcriptional activity, we analysed the accumulation of transcripts
derived from these regions.
To do this, a primer complementary to the 3´ end of the
internal transcribed spacer 2 (ITS2-A) of the 45S rRNA
transcription unit (see Supplementary Fig. S6C) was employed
to generate the cDNA template. The pair 5.8s-Fw/5.8s-Rv
was used to differentially amplify by PCR the unprocessed
rRNA. We observed significantly increased levels of
prerRNA and TE-derived transcripts in pollen RNA from
viroid-infected plants compared to the mock-inoculated
controls (Fig. 5A–C). Similar results were obtained when the
accumulation of both pre-rRNA and TE-derived transcripts
in control and HSVd-infected pollen grains was analysed by
real-time quantitative PCR (Fig. 5D). We thus conclude that
HSVd-infection promotes increased transcriptional activity
of the repetitive DNAs in pollen that were analysed,
revealing that the viroid-induced hypometylation causes changes in
the transcriptional status in cucumber reproductive tissues.
Drastic alterations of the epigenome in response to pathogen
attack have been described for both plants (Agorio and Vera,
2007; Boyko et al., 2007, 2010; Lopez et al., 2011; Dowen
et al., 2012; Luna and Ton, 2012; Yu et al., 2013) and animals
(Tang et al., 2011). These observations support the notion
that epigenetic reprogramming of transcriptional activity is a
general mechanism controlling the host response to pathogen
infection (Zhu et al., 2016). Consistent with this idea, HSVd
accumulation in vegetative tissues causes hypomethylation of
the promoter region of rRNA genes, leading to significant
alterations in rRNA transcription (Martinez et al., 2014;
Castellano et al., 2015). In this study we show that both viroid
mature forms and vd-sRNAs accumulate in pollen grains
of HSVd-infected plants, indicating that during the
pathogenesis process the viroid is able to invade this reproductive
cell. We furthermore demonstrate that general sRNA
profiles are altered in this reproductive tissue; in particular, we
observed increased sRNAs derived from rRNA and TE
transcripts. Increased levels of ribosomal RNA-derived sRNAs
(rb-sRNAs) were previously reported to occur in leaves of
cucumber and N. benthamiana plants infected by HSVd,
reinforcing the close interplay between viroid-induced
pathogenesis and host rRNA metabolism. Importantly, the
observation that sRNAs derived from TEs (te-sRNAs) are also
over-accumulated in infected tissue suggests that the
viroidinduced transcriptional alteration is a more general
phenomenon that is not restricted only to rRNA repeats. In line with
this possibility, bisulfite sequencing clearly correlated HSVd
infection with changes in DNA methylation – in a symmetric
sequence context – in both rDNA and TE.
The hypomethylated status of the rDNA regions analysed
is consistent with the significant size increase of the nucleolus
in the generative nucleus of HSVd-infected pollen. The
nucleolus is mainly composed of active and inactive rDNA (Lam
and Trinkle-Mulcahy, 2015), and consequently it is
reasonable to suppose that changes in nucleolar morphology (Fig
1D) may be associated with alterations in the rRNA
transcriptional activity observed in pollen grains during viroid
infection (Fig. 5). This resembles, at least in part, the
observation that in soybean plants grown at low temperature different
condensation states of nucleololar chromatin (which occurred
in response to the stress) correlated with changes in DNA
methylation levels and transcriptional activity (Stepiński,
2013). Moreover, and considering that the heterochromatin
of the nucleus is mostly occupied by TEs and other
repetitive DNAs (Lippman et al., 2004), it seems likely that HSVd
infection causes a general reduction of DNA methylation at
repeat regions. Interestingly, decondensation of centromeric
regions and rDNA loci also occurs in response to heat stress
(Pecinka et al., 2010), suggesting a general stress response
in heterochromatin. Whether this is connected to increased
metabolic activities that require increased ribosome
production remains to be investigated. Interestingly, we observed
that HSVd-infected pollen had an increased germination rate
compared to pollen from mock-infected plants, which may be
a consequence of increased ribosomal activity.
Together, our data support the idea that HSVd
accumulation in pollen grains promotes host epigenetic alterations, as
evidenced by drastic changes in rDNA and TE expression.
Although the molecular basis of this phenomenon remains
to be fully elucidated, it was recently shown that in infected
leaves HSVd RNA is able to bind and functionally subvert host
HDA6, promoting rDNA hypomethylation (Castellano et al.,
2016). HDA6 acts as an epigenetic regulator required to confer
memory of the silent state and to maintain DNA methylation
(Aufsatz et al., 2002; Probst et al., 2004; Earley et al., 2010; Liu
et al., 2012; Blevins et al., 2014; Hristova et al., 2015). As we
observed similar changes in DNA methylation in pollen and
vegetative tissues, this suggests that HSVd also impairs the
function of HDA6 in pollen. However, in contrast to
HSVdinfected cucumber leaves where 21-nt and 24-nt rb-sRNAs
were respectively up- and down-regulated, the methylation
loss in pollen grains was accompanied by a general increase of
sRNAs in all size classes. An increase of 21-nt TE or
rRNAderived sRNAs is a known characteristic of Pol II
transcription of repetitive regions upon down-regulation of epigenetic
factors such as DDM1 or HDA6 (Earley et al., 2010; McCue
et al., 2012). Consequently, our results suggest that although
the altered epigenetic scenario is similar in both vegetative and
reproductive tissues, specific aspects of the HDA6-dependent
regulatory mechanisms underlying this phenomenon differ. In
this regard, recent data have demonstrated that DNA
methylation can also be modulated by DCL-independent siRNAs
(Yang et al., 2016; Dalakouras et al., 2016; Ye et al., 2016).
These siRNAs (ranging from 20 to 60 nt in length) are mainly
derived from repetitive sequences and loaded in AGO4 to
direct DNA methylation (Ye et al., 2016). Further studies are
needed in order to determine if this non-canonical regulatory
pathway could be also affected in HSVd-infected pollen grains.
Furthermore, it will be important to test whether
epigenome changes in response to HSVd infection have
functional consequences in the next generation of plants. Thus
far, transgenerational effects of stress are rather questionable
(Pecinka and Scheid, 2012); however, our data show a stress
response affecting the chromatin status of the generative
nucleus, suggesting that these changes could potentially be
inherited to the next generation.
In summary, we have shown that HSVd infection induces
hypomethylation of rRNA genes and TEs in pollen grains,
providing the first example of epigenome changes in
reproductive cells upon pathogen infection.
Supplementary data are available at JXB online.
Figure S1. Validation of HSVd-infected cucumber plants.
Figure S2. PCR of total RNA extracts in order to discard
Figure S3. Analysis of differentially expressed
ribosomalderived sRNAs in infected pollen.
Figure S4. Analysis of TE-derived sRNAs differentially
expressed in infected cucumber pollen.
Figure S5. Validation by stem-loop qRT-PCR of
representative sRNAs highly accumulated in HSVd-infected
Figure S6. Diagram of the rDNA intergenic region
analysed by bisulfite sequencing.
Figure S7. HSVd infection affects the methylation patterns
of TE DNA in cucumber leaves.
Table S1. Description of primers used in the qRT-PCR
assays of rRNA and TE transcripts.
Table S2. Description of primers used in stem-loop
The authors thank Dr A. Monforte for help in the collection of pollen grains.
This work was supported by grants AGL2013-47886-R and
BIO2014-61826EXP (GG) and BIO2014-54862-R (VP) from the Spanish Granting Agency
(Direccion General Investigacion Cientifica). MC was the recipient of a PhD
fellowship from the Ministerio de Educación, Cultura y Deportes of Spain.
Author contributions were as follows. MC performed the experiments,
discussed the results, prepared figures and revised the manuscript. GM
performed the experiments, discussed the results, prepared figures and revised
the manuscript. MCM collaborated in the qRT-PCR assays. JR collaborated
in the confocal analysis. CK and VP discussed the results and revised the
main manuscript text. GG designed the experiments, discussed the results,
prepared figures and wrote the main manuscript text.
Adkar-Purushothama CR, Brosseau C, Giguere T, Sano T, Moffett
P, Perreault JP. 2015. Small RNA derived from the virulence modulating
region of the potato spindle tuber viroid silences callose synthase genes of
tomato plants. Plant Cell 27, 2178–2194.
Agorio A, Vera P. 2007. ARGONAUTE4 is required for resistance to
Pseudomonas syringae in Arabidopsis. Plant Cell 19, 3778–3790.
Alvarez ME, Nota F, Cambiagno DA. 2010. Epigenetic control of plant
immunity. Molecular Plant Pathology 11, 563–576.
Aparicio F, Sanchez-Pina MA, Sanchez-Navarro JA, Pallas V.
1999. Location of prunus necrotic ringspot ilarvirus within pollen grains
of infected nectarine trees: evidence from RT-PCR, dot-blot and in situ
hybridisation. European Journal of Plant Pathology 105, 623–627.
Aufsatz W, Mette MF, van der Winden J, Matzke M, Matzke AJM.
2002. HDA6, a putative histone deacetylase needed to enhance DNA
methylation induced by double-stranded RNA. Embo Journal 21,
Barba M, Ragozzino E, Faggioli F. 2007. Pollen transmission of Peach
latent mosaic viroid. Journal of Plant Pathology 89, 287–289.
Blevins T, Pontvianne F, Cocklin R, et al. 2014. A two-step process for
epigenetic inheritance in Arabidopsis. Molecular Cell 54, 30–42.
Boyko A, Blevins T, Yao Y, Golubov A, Bilichak A, Ilnytskyy Y,
Hollunder J, Meins F, Jr, Kovalchuk I. 2010. Transgenerational
adaptation of Arabidopsis to stress requires DNA methylation and the
function of Dicer-like proteins. PLoS ONE 5, e9514.
Boyko A, Kathiria P, Zemp FJ, Yao Y, Pogribny I, Kovalchuk I. 2007.
Transgenerational changes in the genome stability and methylation in
pathogen-infected plants: (virus-induced plant genome instability). Nucleic
Acids Research 35, 1714–1725.
Boyko A, Kovalchuk I. 2011. Genetic and epigenetic effects of plant–
pathogen interactions: an evolutionary perspective. Molecular Plant 4,
Cao MJ, Du P, Wang XB, Yu YQ, Qiu YH, Li WX, Gal-On A,
Zhou CY, Li Y, Ding SW. 2014. Virus infection triggers widespread
silencing of host genes by a distinct class of endogenous siRNAs in
Arabidopsis. Proceedings of the National Academy of Sciences, USA 111,
Card SD, Pearson MN, Clover GRG. 2007. Plant pathogens transmitted
by pollen. Australasian Plant Pathology 36, 455–461.
Castellano M, Martinez G, Pallás V, Gómez G. 2015. Alterations
in host DNA methylation in response to constitutive expression of Hop
stunt viroid RNA in Nicotiana benthamiana plants. Plant Pathology 64,
Castellano M, Pallás V, Gómez G. 2016. A pathogenic long non-coding
RNA redesigns the epigenetic landscape of the infected cells by subverting
host Histone Deacetylase 6 activity. New Phytologist 211, 1311–1322.
Czimmerer Z, Hulvely J, Simandi Z, et al. 2013. A versatile method
to design stem-loop primer-based quantitative PCR assays for detecting
small regulatory RNA molecules. PLoS ONE 8, e55168.
Dalakouras A, Dadami E, Wassenegger M, Krczal G, Wassenegger
M. 2016. RNA-directed DNA methylation efficiency depends on trigger
and target sequence identity. Plant Journal 87, 202–214.
Dellaporta SL, Wood J, Hicks JB. 1983. A plant DNA minipreparation:
version II. Plant Molecular Biology Reporter 1, 19–21.
Ding B. 2009. The biology of viroid–host interactions. Annual Review of
Phytopathology 47, 105–131.
Dowen RH, Pelizzola M, Schmitz RJ, Lister R, Dowen JM, Nery
JR, Dixon JE, Ecker JR. 2012. Widespread dynamic DNA methylation
in response to biotic stress. Proceedings of the National Academy of
Sciences, USA 109, E2183–E2191.
Eamens AL, Smith NA, Dennis ES, Wassenegger M, Wang MB.
2014. In Nicotiana species, an artificial microRNA corresponding to
the virulence modulating region of Potato spindle tuber viroid directs
RNA silencing of a soluble inorganic pyrophosphatase gene and the
development of abnormal phenotypes. Virology 450, 266–277.
accompanied by short RNA fragments that are characteristic of
posttranscriptional gene silencing. Nucleic Acids Research 29, 2395–2400.
Earley KW , Pontvianne F , Wierzbicki AT , Blevins T , Tucker S , CostaNunes P , Pontes O , Pikaard CS . 2010 . Mechanisms of HDA6-mediated rRNA gene silencing: suppression of intergenic Pol II transcription and differential effects on maintenance versus siRNA-directed cytosine methylation . Genes & Development 24 , 1119 - 1132 .
Flores R , Hernandez C , Martinez de Alba AE , Daros JA , Di Serio F. 2005 . Viroids and viroid-host interactions . Annual Review of Phytopathology 43 , 117 - 139 .
Garcia JA , Pallas V. 2015 . Viral factors involved in plant pathogenesis .
Current Opinion in Virology 11, 21 - 30 .
Gomez G , Martinez G , Pallas V. 2008 . Viroid-induced symptoms in Nicotiana benthamiana plants are dependent on RDR6 activity . Plant Physiology 148 , 414 - 423 .
Gomez G , Martinez G , Pallas V. 2009 . Interplay between viroid-induced pathogenesis and RNA silencing pathways . Trends in Plant Science 14 , 264 - 269 .
Gomez G , Pallas V. 2006 . Hop stunt viroid is processed and translocated in transgenic Nicotiana benthamiana plants . Molecular Plant Pathology 7 , 511 - 517 .
Gomez G , Pallas V. 2013 . Viroids: a light in the darkness of the lncRNAdirected regulatory networks in plants . New Phytologist 198 , 10 - 15 .
Hristova E , Fal K , Klemme L , Windels D , Bucher E. 2015 . HISTONE DEACETYLASE6 controls gene expression patterning and DNA methylation-independent euchromatic silencing . Plant Physiology 168 , 1298 - 1308 .
Kryczyński S , Paduch-Cichal E , Skrzeczkowski LJ. 1988 .
Journal of Phytopathology 121 , 51 - 57 .
Lam YW , Trinkle-Mulcahy L. 2015 . New insights into nucleolar structure and function . F1000Prime Reports 7 , 48 .
Law JA , Jacobsen SE . 2010 . Establishing, maintaining and modifying DNA methylation patterns in plants and animals . Nature Reviews Genetics 11 , 204 - 220 .
Li LC , Dahiya R . 2002 . MethPrimer: designing primers for methylation PCRs . Bioinformatics 18 , 1427 - 1431 .
Li Z , Zhang ZH , Yan PC , Huang SW , Fei ZJ , Lin K. 2011 . RNA-Seq improves annotation of protein-coding genes in the cucumber genome .
BMC Genomics 12 , 540 .
Lippman Z , Gendrel AV , Black M , et al. 2004 . Role of transposable elements in heterochromatin and epigenetic control . Nature 430 , 471 - 476 .
2012. HDA6 directly interacts with DNA methyltransferase MET1 and maintains transposable element silencing in Arabidopsis . Plant Physiology 158 , 119 - 129 .
Lopez A , Ramirez V , Garcia-Andrade J , Flors V , Vera P. 2011 . The RNA silencing enzyme RNA polymerase V is required for plant immunity .
PLoS Genetics 7 , e1002434 .
Luna E , Ton J. 2012 . The epigenetic machinery controlling transgenerational systemic acquired resistance . Plant Signaling & Behavior 7 , 615 - 618 .
Martinez G , Castellano M , Tortosa M , Pallas V , Gomez G. 2014 .
A pathogenic non-coding RNA induces changes in dynamic DNA methylation of ribosomal RNA genes in host plants . Nucleic Acids Research 42 , 1553 - 1562 .
Martinez G , Donaire L , Llave C , Pallas V , Gomez G. 2010 . Highthroughput sequencing of Hop stunt viroid-derived small RNAs from cucumber leaves and phloem . Molecular Plant Pathology 11 , 347 - 359 .
Martinez G , Pallas V , Gomez G. 2008 . Analysis of symptoms developed in Nicotiana benthamiana plants expressing dimeric forms of Hop stunt viroid . Journal of Plant Pathology 90 , 121 - 124 .
Martinez G , Slotkin RK . 2012 . Developmental relaxation of transposable element silencing in plants: functional or byproduct? Current Opinions in Plant Biology 15 , 496 - 502 .
Matsushita Y , Tsuda S. 2014 . Distribution of Potato spindle tuber viroid in reproductive organs of Petunia during its developmental stages .
Phytopathology 104 , 964 - 969 .
McCue AD , Nuthikattu S , Reeder SH , Slotkin RK . 2012 . Gene expression and stress response mediated by the epigenetic regulation of a transposable element small RNA . PLoS Genetics 8 , e1002474 .
Mercer TR , Mattick JS . 2013 . Structure and function of long noncoding RNAs in epigenetic regulation . Nature Structural & Molecular Biology 20 , 300 - 307 .
Navarro B , Gisel A , Rodio ME , Delgado S , Flores R , Di Serio F. 2012 .
Biochimie 94 , 1474 - 1480 .
Pallas V , Gomez G , Amari K , Cañizares MC , Candresse T. 2003 . Hop stunt viroid in apricot and almond . In: Hadidi A, Randles J , Semancik J , Flores R , eds. Viroids. St Paul, MN, USA: Science Publishers, 168 - 170 .
Papaefthimiou I , Hamilton A , Denti M , Baulcombe D , Tsagris M , Tabler M. 2001 . Replicating potato spindle tuber viroid RNA is Pecinka A , Dinh HQ , Baubec T , Rosa M , Lettner N , Scheid OM .
2010. Epigenetic regulation of repetitive elements is attenuated by prolonged heat stress in Arabidopsis . Plant Cell 22 , 3118 - 3129 .
Pecinka A , Scheid OM . 2012 . Stress-induced chromatin changes: a critical view on their heritability . Plant Cell Physiology 53 , 801 - 808 .
Probst AV , Fagard M , Proux F , et al. 2004 . Arabidopsis histone deacetylase HDA6 is required for maintenance of transcriptional gene silencing and determines nuclear organization of rDNA repeats . Plant Cell 16 , 1021 - 1034 .
Rodriguez-Negrete E , Lozano-Duran R , Piedra-Aguilera A , Cruzado L , Bejarano ER , Castillo AG . 2013 . Geminivirus Rep protein interferes with the plant DNA methylation machinery and suppresses transcriptional gene silencing . New Phytologist 199 , 464 - 475 .
Ruiz-Ferrer V , Voinnet O. 2009 . Roles of plant small RNAs in biotic stress responses . Annual Review of Plant Biology 60 , 485 - 510 .
Sano T. 2003 . Hop stunt viroid in cucumber . In: Hadidi A, Randles J , Semancik J , Flores R , eds. Viroids. St Paul, MN, USA: Science Publishers, 134 - 136 .
Sha AH , Lin XH , Huang JB , Zhang DP . 2005 . Analysis of DNA methylation related to rice adult plant resistance to bacterial blight based on methylation-sensitive AFLP (MSAP) analysis . Molecular Genetics & Genomics 273 , 484 - 490 .
Shimura H , Pantaleo V , Ishihara T , Myojo N , Inaba J , Sueda K , Burgyan J , Masuta C. 2011 . A viral satellite RNA induces yellow symptoms on tobacco by targeting a gene involved in chlorophyll biosynthesis using the RNA silencing machinery . PLoS Pathogens 7 , e1002021 .
Singh RP . 1970 . Seed transmission of potato spindle tuber virus in tomato and potato . American Potato Journal 47 , 225 - 227 .
Singh RP , Boucher A , Somerville TH . 1993 . Interactions between a mild and a severe strain of potato spindle tuber viroid in doubly infected potato plants . American Potato Journal 70 , 85 - 92 .
Singh RP , Ready KF , Nie X. 2003 . Biology . In: Hadidi A, Randles J , Semancik J , Flores R , eds. Viroids. St Paul, MN, USA: Science Publishers, 30 - 48 .
Slotkin RK , Martienssen R . 2007 . Transposable elements and the epigenetic regulation of the genome . Nature Reviews Genetics 8 , 272 - 285 .
Smith NA , Eamens AL , Wang MB . 2011 . Viral small interfering RNAs target host genes to mediate disease symptoms in plants . PLoS Pathogens 7 , e1002022 .
Stępiński D. 2013 . Nucleolar chromatin organization at different activities of soybean root meristematic cell nucleoli . Protoplasma 250 , 723 - 730 .
Tang B , Zhao R , Sun Y , Zhu Y , Zhong J , Zhao G , Zhu N. 2011 .
Interleukin-6 expression was regulated by epigenetic mechanisms in response to influenza virus infection or dsRNA treatment . Molecular Immunology 48 , 1001 - 1008 .
2007. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs . Plant Methods 3 , 12 .
Wah YFWC , Symons RH . 1999 . Transmission of viroids via grape seeds .
Journal of Phytopathology 147 , 285 - 291 .
Wang MB , Bian XY , Wu LM , et al. 2004 . On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites . Proceedings of the National Academy of Sciences , USA 101 , 3275 - 3280 .
Ye R , Chen Z , Lian B , Rowley MJ , Xia N , Chai J , Li Y , He XJ , Wierzbicki AT , Qi Y. 2016 . A Dicer-independent route for biogenesis of siRNAs that direct DNA methylation in Arabidopsis . Molecular Cell 61 , 222 - 235 .
2016. Dicer-independent RNA-directed DNA methylation in Arabidopsis.
Cell Research 26 , 66 - 82 .
Yu A , Lepere G , Jay F , et al. 2013 . Dynamics and biological relevance of DNA demethylation in Arabidopsis antibacterial defense . Proceedings of the National Academy of Sciences , USA 110 , 2389 - 2394 .
Zhu QH , Shan WX , Ayliffe MA , Wang MB . 2016 . Epigenetic mechanisms: an emerging player in plant-microbe interactions . Molecular Plant-Microbe Interactions 29 , 187 - 196 .