Download MERE1, a Low-Copy-Number Copia-Type

MERE1, a Low-Copy-Number Copia-Type Retroelement
in Medicago truncatula Active during Tissue Culture1[C][W]
Alexandra Rakocevic, Samuel Mondy, Leı̈la Tirichine, Viviane Cosson, Lysiane Brocard, Anelia Iantcheva,
Anne Cayrel, Benjamin Devier, Ghada Ahmed Abu El-Heba2, and Pascal Ratet*
Institut des Sciences du Végétal, CNRS, 91198 Gif sur Yvette, France (A.R., S.M., L.T., V.C., L.B., A.C., B.D.,
G.A.A.E.-H., P.R.); and AgroBioinstitute, 1164 Sofia, Bulgaria (A.I.)
We have identified an active Medicago truncatula copia-like retroelement called Medicago RetroElement1-1 (MERE1-1) as an
insertion in the symbiotic NSP2 gene. MERE1-1 belongs to a low-copy-number family in the sequenced Medicago genome.
These copies are highly related, but only three of them have a complete coding region and polymorphism exists between the
long terminal repeats of these different copies. This retroelement family is present in all M. truncatula ecotypes tested but also
in other legume species like Lotus japonicus. It is active only during tissue culture in both R108 and Jemalong Medicago
accessions and inserts preferentially in genes.
Transposable elements are mobile genetic elements
present in a wide range of organisms from bacteria to
eukaryotes and are usually classified in two different
groups. Class I elements, called retrotransposons,
transpose through an RNA intermediate that is reverse
transcribed into a linear double-stranded DNA before
its integration into the host genome (copy-and-paste
mechanism). Class II elements, called DNA transposons, transpose directly via a DNA intermediate
(cut-and-paste mechanism). The replicative mode of
transposition of class I retrotransposons may rapidly
increase their copy number, which can be extremely
high in eukaryote genomes.
Because they can invade genomes, minimizing their
copy number is particularly important to maintain the
genome integrity, and active retroelements are generally found in low-copy-number families (Madsen
et al., 2005). These elements are present in plants and
mammals, but long terminal repeat (LTR) transposons
appear to be rare in the latter. LINEs, SINEs, Ty31
This work was supported by a fellowship from the French
Ministère de l’Education Nationale, de l’Enseignement Supérieur et
de la Recherche, to A.R., by the Grain Legumes Integrated Project
(grant no. FOOD–CT–2004–506223 to L.B. and A.C.), and by a
UNESCO-L’OREAL-cosponsored Fellowship for Young Women in
Life Science-2006 to G.A.A.E.-H.
2
Present address: Agricultural Genetic Engineering Research
Institute, Agricultural Research Centre, 9 Gamaa St., Giza 12619,
Egypt.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Pascal Ratet ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.109.138024
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gypsy, and Ty1-copia appear to be abundant in plants
(Kumar and Bennetzen, 1999). Transposable elements
are usually located in intergenic regions and tend to
accumulate in centromeres, telomeres, and heterochromatic regions. An analysis of 233 Mb of the Medicago
truncatula genome shows that retroelements constitute
about 9.6% of the currently available genomic sequence, and the majority of the LTR retroelements
belong to either the copia or gypsy superfamily (Wang
and Liu, 2008).
Despite the fact that they are widespread and
abundant, only a few of them have been described as
active in plants: Tnt1 (Grandbastien et al., 1989), Tto1
(Hirochika et al., 2000), Tos17 (Hirochika, 1997), and
LORE1 and LORE2 (Fukai et al., 2008). New transposition events in genomes have been noticed, but the
correlation between transcription induction and transposition is not always obvious. Plant retroelements can
be activated by tissue culture in tobacco (Nicotiana
tabacum [Tto1]; Hirochika, 1993), rice (Oryza sativa
[Tos17]; Hirochika et al., 1996; Miyao et al., 2003), and
M. truncatula (Tnt1; d’Erfurth et al., 2003). Plant retroelements are also activated by wounding or pathogen
attack (Mhiri et al., 1997; Takeda et al., 1999; Melayah
et al., 2001). For other plant mobile retroelements, the
activation conditions have not been established
(LORE1; Madsen et al., 2005). The main characteristic
of these retroelements is their ability to insert randomly in the genome and to alter gene function
following disruption. The creation of this genetic variability might play an important role as a source of
genome plasticity needed for plant evolution (Wessler
et al., 1995).
Retroelements carry in their LTR region, which can
be subdivided into U3, R, and U5 regions, cis-acting
sequences that control their expression in the host
(Kumar and Bennetzen, 1999). Specific sequences in
the LTR U3 regions have been shown to be involved in
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A Mobile Retroelement in Medicago truncatula
the induction of the retroelement transcription by
various biotic and abiotic stress factors (Casacuberta
and Grandbastien, 1993; Vernhettes et al., 1997; Takeda
et al., 1999). The variability observed in these U3
regions among the different subfamilies of Tnt1 is
thought to be associated with the ability of these
promoters to respond to different signaling stress
molecules (Vernhettes et al., 1997; Araujo et al., 2001;
Beguiristain et al., 2001). DNA methylation seems to
be an important factor controlling retroelement expression in plants (Martienssen and Colot, 2001), because high levels of cytosine methylation have been
associated with transcriptional inactivity. This control
process is apparently different between mammals and
plants. Repetitive elements are methylated in both
organisms, but whereas most mammalian exons are
methylated, plant exons are generally not. Thus, targeting methylation specifically to transposons appears
to be restricted to plants (Rabinowicz et al., 2003).
Transcription reduction of Tto1, Ttnt1, and Tos17 was
correlated to their methylation status in Arabidopsis
(Arabidopsis thaliana) and rice (Hirochika et al., 2000;
Liu et al., 2004; Perez-Hormaeche et al., 2008), but the
factors controlling this methylation are not clearly
understood. Moreover, posttranscriptional regulation
of the transposition is also an important step of the
retrotransposition. In Neurospora crassa, the LINE1-like
retroelement is repressed by the posttranscriptional
gene silencing machinery independently of DNA
methylation (Nolan et al., 2005). In contrast, the
DNA methylation machinery had no obvious effects
on its expression. In Saccharomyces cerevisiae, Ty1 copy
number control occurs by both transcriptional and
posttranscriptional cosuppression (Garfinkel et al.,
2003).
We describe here Medicago RetroElement1-1 (MERE11), an active retroelement in M. truncatula. This retroelement belongs to a small copia-like retroelement
family of five to 10 members corresponding to the
Mtr16 family defined by Wang and Liu (2008). This
family is also present in other legumes. MERE1-1 has
transposed in several M. truncatula mutant collections,
and its transposition activity is correlated to in vitro
tissue culture and to methylation of its sequence.
Identification of MERE1, an Active Retroelement
in M. truncatula
Figure 1. Phenotype of the nsp2 nonnodulating mutant. A and B, Wildtype (A) and nsp2-4 (B) plants inoculated with S. meliloti FSM.Ma
strain. C, Light microscopy of wild-type noninoculated root hairs. D
and E, Curled wild-type root hairs or forming “shepherd’s crook”
observed after inoculation with Rm1021 strain. F, Nonnoculated root
hair in MtNSP2. G, Noncurling MtNSP2 root hair inoculated with
Rm1021 strain. [See online article for color version of this figure.]
During a screen for M. truncatula nodulation mutants in a T-DNA mutant collection (Scholte et al.,
2002), a nonnodulating (nod2) mutant (ms219) was
identified (Brocard et al., 2006). We have shown that
the corresponding (somaclonal) mutation does not
correlate with the presence of the T-DNA in this
mutant line. No root hair deformation or primordium
formation was observed after inoculation of the mutant with Sinorhizobium meliloti, indicating that the
symbiotic process was blocked early (Fig. 1), similarly
to the nfp mutant (Amor et al., 2003). The expression of
marker genes for early nodule development, ENOD11
(Journet et al., 2001), ENOD12 (Journet et al., 1994),
and MtN6 and Rip1 (Cook et al., 1995), was absent
(ENOD11 and ENOD12) or reduced (MtN6 and Rip1)
in the mutant, in agreement with an alteration of Nod
factor perception (Supplemental Fig. S1). Allelic tests
performed between ms219 and other mutants affected
in early Nod factor signaling, nfp (Amor et al., 2003),
RESULTS
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Rakocevic et al.
dmi1, dmi2, and dmi3 (Catoira et al., 2000), indicated
that these genes were not affected in ms219 (data not
shown). Because the phenotype and the allelic tests
indicated that the mutation could correspond to a new
symbiotic locus, the mutation was mapped using
crosses between the ms219 mutant (background
R108) and the Jemalong 5 (J5) line. The F2 populations
from six independent crosses were used for mapping
using markers based on PCR fragment-length polymorphisms (see “Materials and Methods”; Supplemental Table S1). The mutation was located to a
3-centimorgan region on chromosome 3 bordered
by bacterial artificial chromosomes AC149303 and
AC126782 (www.medicago.org). This genomic region
includes the NSP2 gene (Kalo et al., 2005). Therefore,
this gene was considered as a candidate for the symbiotic gene despite the different phenotype described
for the known nsp2 mutant (root hair branching).
Amplification of the NSP2 coding sequence in three
independent ms219 plants using long-range DNA
polymerase demonstrated that the NSP2 sequence
contained an insert of approximately 5.3 kb, which
was likely responsible for the mutation (data not
shown). Sequencing of the insert identified a retrotransposon, inserted at base pair 1,062 (Fig. 2A) of the
NSP2 coding sequence. We named this transposon
MERE1-1.
To demonstrate that the insertion of MERE1-1 into
the NSP2 gene cosegregated with the mutant phenotype, the wild type and the nsp2 mutant allele, named
nsp2-4 hereafter, were PCR amplified using primers
specific for both alleles (Fig. 2B). This showed that the
nsp2-4::MERE1-1 insertion cosegregated with the nod2
phenotype. Finally, to prove that the insertion in the
NSP2 gene is responsible for the phenotype, the nsp2-4
allele was crossed with the nsp2-1 allele (Kalo et al.,
2005). The F1 individuals had a nod2 phenotype,
demonstrating the allelism of both mutants.
MERE1-1 Belongs to a Small Family of
Copia Retroelements
The MERE1-1 retroelement identified at the NSP2
locus is a 5,300-bp-long element and has two long
terminal repeat (LTR) regions of 574 bp (Fig. 3A). This
element is flanked by a 5-bp duplication of host DNA
corresponding to the target site duplication (TSD). A
single 1,305-amino acid open reading frame (ORF),
starting at base pair 746 and ending at base pair 4,660,
displays, after in silico translation, a strong similarity
with the GAG-POL protein of plant retroviral elements. The location of the reverse transcriptase domain behind the endonuclease (endo) classifies
MERE1 as a Ty1-copia retrotransposon.
BLAST searches against the M. truncatula Jemalong
genome sequence allowed the identification of five
complete MERE1 elements, numbered MERE1-1 to
MERE1-5, corresponding to the Mtr16 family recently
described by Wang and Liu (2008; for accession numbers, see “Materials and Methods”). MERE1-1 has
been identified as the element present at the NSP2
locus. Three of the MERE1 elements have the 1,305amino acid full-length ORF (MERE1-1, MERE1-3, and
MERE1-4). The MERE1-2 sequence has an additional T
at nucleotide 2,852, creating a frame shift generating a
truncated 707-amino acid ORF. The MERE1-5 sequence
contains a stop codon at position 1,950, resulting in a
truncated 425-amino acid ORF. The three full-length
Figure 2. Characterization of the nsp2-4 mutant. A, Structure of the NSP2 gene. The locations of the NSP2 GRAS domain (gray
box) and variable N-terminal region (black box) are indicated. Positions are given in base pairs relative to the AUG. MERE1-1 is
inserted at base pair 1,062 in the variable N-terminal region (3#–5# direction). nsp2-1 and nsp2-2 are two fast-neutron-generated
mutants containing a 435-bp in-frame deletion. nsp2-3 is an ethylmethane sulfonate mutant allele. B, Cosegregation of the
MERE1 insertion in the NSP2 locus (NSP2-4::MERE1) with the nonnodulating phenotype of nsp2-4. A total of 41 F2 nod2 and 96
F2 nod+ plants from a backcross between R108 NSP2-4 and J5 NSP2 were used to PCR amplify wild-type or mutant loci (the
results for 10 nod2 and 10 nod+ plants are presented). The absence of amplification products using NSP2-specific primers (NSP25#/NSP2-3#) indicates that all nod2 individuals are homozygous for the NSP2-4::MERE1 insertion. Amplification of a NSP2-3#/
MERE1-specific product using primers NSP2-3# and MERE1 in the same samples shows the presence of the MERE1 insertion. The
nod+ individuals are either wild type (no NSP2-MERE1 product) or heterozygous (presence of the two PCR products) for the
NSP2-4::MERE1 insertion.
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A Mobile Retroelement in Medicago truncatula
Figure 3. Structure of the MERE1-1 retroelement representing the MERE1 family. A, Schematic representation of MERE1-1. Gray
boxes at the two extremities represent LTRs, divided into three parts: U3 (promoter), R (polyadenylation signal), and U5. Shaded
boxes indicate regions showing homology with characteristic retrotransposon proteins. The hatched box below the RT region
represents the DNA fragment used as a probe in Southern- and northern-blot analyses. From N to C terminus, the unique MERE1-1
ORF shows extensive amino acid homology to copia GAG (structural core proteins), PROT (protease involved in maturation of
GAG polyproteins), ENDO (endonuclease involved in integration into the host DNA) and RT (reverse transcriptase) domains.
Putative primer binding sites (PBS) and polypurine tract (PPT), necessary for cDNA synthesis of the retrotransposon, are shown
below, as well as the complementary region between PBS and the 3# end of legume tRNAmet. B, Sequence comparison of the LTR
regions from MERE1-1 to MERE1-5. The U5 region shows a high level of identity between the five sequences, whereas the U3
region shows significant variability. The putative TATA box is indicated by the box. The positions of the different oligonucleotides
used in this study are indicated by arrows above the sequences. MERE6N and MERE5N represent the positions of the specific
oligonucleotides MERE61, MERE62, MERE63, MERE64, MERE51, and MERE52 (for sequences, see “Materials and Methods”).
[See online article for color version of this figure.]
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Rakocevic et al.
ORF elements (MERE1-1, -3, and -4) could represent
active retroelements, and the two others could represent inactive ones. Comparison of the MERE1 family
LTR sequences shows conservation of the U5 region
but many differences in the U3 promoter region (Fig.
3B). Among the five elements described above,
MERE1-1, -2, and -3 have identical 5# and 3# LTRs.
The two LTRs from MERE1-4 show 3-bp differences,
and MERE1-5 LTRs show 6-bp differences. BLASTn
search analysis indicated the presence of at least five
solo LTRs related to the MERE1 family in the Medicago
pseudogenome. Phylogenetical analysis indicated that
none of these correspond to the LTRs of the MERE1-1
to MERE1-5 elements (Supplemental Fig. S2).
The MERE1 Element Is Present at Low Copy Number in
M. truncatula and Other Legume Species
To evaluate the copy number of the MERE1 element
in the entire Medicago genome, a Southern-blot analysis was performed using a probe corresponding to the
3# extremity of the polymerase region (Fig. 3A). This
analysis indicates that the MERE1 family is present at
moderate copy number in the different lines analyzed
(Fig. 4A), suggesting that only a few additional copies
are present in the heterochromatic unsequenced re-
gion of the genome. To analyze the MERE1 family
members in more detail, a retrotransposon display
method (see “Materials and Methods”) was used,
which allowed a better discrimination of the different
MERE1 elements present in the genome. Figure 4B
shows that many bands are common to J5 and the
related 2HA line and to R108 and its derivative nsp2-4.
However, a polymorphism is detected between the
Jemalong and R108 lines. Similarly, using this technique, a polymorphism can be detected between the
different lines from a small M. truncatula ecotype
collection (Fig. 4C), despite a rather constant copy
number of the element in these different lines. Bands
shared by some of these ecotypes (e.g. F83.005.9,
DZA0517, F20061A, F34024A, and ESP158A) were
identified, probably indicating a common geographical origin.
Data mining also indicated that MERE1-1 belongs to
clade 1 (Wang and Liu, 2008) of copia plant retroelements and that MERE1-like retroelements are present
in different legume species (Supplemental Fig. S3).
The MERE1 family is related to the Cajanus Panzee
element described by Lall et al. (2002), and we could
identify at least two full-length retroelements with
LTR sequences similar to MERE1 in the Lotus japonicus
MG.20 genome (AP004536 and AP006407). The LTR
Figure 4. Copy number of the MERE1 retroelement in M. truncatula. A, Southern-blot analysis of M. truncatula 2HA, J5, R108,
and NSP2-4 lines. Ten micrograms of gDNA was double digested with HpaI and NcoI restriction enzymes. The probe used is
indicated in Figure 3A. B, Transposon display of MERE1 5# insertion sites in wild-type and NSP2-4 mutant lines. The fragments
labeled with asterisks represent copies of the MERE1-1 element different between 2HA and J5 lines. DNA was digested with the
AseI restriction enzyme. PCR amplification was done using the oligonucleotide pairs MERE2/Ase1 and MERE1/Ase2. C,
Transposon display of MERE1-1 insertion sites in different Medicago accessions. Arabidopsis (ecotype Columbia [Col0]) was
used as a negative control for this experiment. The average number of retroelements in the different ecotypes is less than 10. The
origin of the different Medicago lines is given in “Materials and Methods.” DNA was digested with the AseI restriction enzyme.
PCR amplification was done using the oligonucleotide pairs MERE2/Ase1 and MERE1/Ase2.
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A Mobile Retroelement in Medicago truncatula
sequences from these two elements are 65% similar,
and only one L. japonicus element has a complete
GAG-POL protein. The analysis also indicates that
closely related elements are present in Populus tremulus
and Ipomoea batatas plants.
MERE1 Transposes in in Vitro-Produced
M. truncatula Plants
Because the MERE1 copy number is low and seems
to be stable in wild-type Medicago plants, we hypothesized that transposition at the NSP2 locus was induced by the tissue culture treatment in our Medicago
transgenic plants. Thus, in order to rule out that the
transposition of the MERE1 element was restricted to
the ms219 nod2 mutant, the presence of MERE1 additional copies was monitored in transgenic plants of
different mutant collections generated by in vitro culture in our laboratory.
MERE1 transposition was first tested using two
Medicago R108 collections: the Tnt1 R108 collection
(Tnk mutants, 250 lines; d’Erfurth et al., 2003) and the
T-DNA collection (GKB mutants, 500 lines; Scholte
et al., 2002; Brocard et al., 2006). The different MERE1
borders produced in these experiments suggest that
transposition occurred in some of the mutant lines
(Fig. 5, A and B), because the pattern of fragments
observed for these plants is unique. Altogether, this
experiment demonstrates that transposition occurred
in 73 plants over a total of 183 analyzed plants (40% of
plants with new transposition events). This represents
one to more than 10 new insertions in the regenerated
plants and an average of one new copy per plant in
these collections.
MERE1 activity was also tested in regenerated
Jemalong 2HA plants (Tnt1 insertion mutant collection). As the LTRs of the different MERE1 members
present in the Jemalong genome have differences in
Figure 5. Transposon display of
MERE1 elements in in vitro-regenerated plants. DNA was digested
with the AseI restriction enzyme.
The additional fragments detected
in lines marked with asterisks correspond to new insertion sites of
MERE1. Bands larger than 1,500 bp
are PCR experimental artifacts. A,
Tnk23 regenerated plants from
the Tnt1 collection (R108 background); five out of 23 analyzed
plants are shown. PCR amplification was done using the oligonucleotide pairs MERE51+MERE52/
Ase1 and MERE61/Ase2. B, GKB
regenerated plants from the T-DNA
collection (R108 background); 16
out of 39 analyzed plants are
shown. PCR amplification was
done using the oligonucleotide
pairs MERE51+MERE52/Ase1 and
MERE61/Ase2. C and D, Transposition of the various MERE1 elements
in regenerated 2HA Jemalong
plants. The analysis of eight out of
96 lines is shown. PCR amplification was done using the oligonucleotide pairs MERE51+MERE52/Ase1
for PCR1 followed by MERE61/Ase2
for MERE1-1, MERE62/Ase2 for
MERE1-2/MERE1-3, MERE63/Ase2
for MERE1-4, and MERE64/Ase2
for MERE1-5 for PCR2.
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Rakocevic et al.
the 3# extremity, specific primers were designed to
allow the specific amplification of the different transposon borders (MERE1-2 and MERE1-3 cannot be
discriminated by this technique). This experiment
(Fig. 5, C and D) shows that new MERE1-specific
fragments could be detected in some of the transgenic
plants, indicating that transposition of MERE1 occurs
in a subset of the regenerated plants from this collection. Surprisingly, we were able to detect the presence
of new copies only for the MERE1-1 element, indicating that it could be the only active/mobile retroelement during tissue culture in Medicago.
PCR amplification of the MERE1-specific borders in
a larger number of regenerated Jemalong 2HA and
R108 plants confirmed that MERE1-1 is indeed the
only active retroelement in these two plants (data not
shown). In addition, this analysis indicates that in the
two Medicago lines transposition is restricted to tissue
culture, because no new copies could be detected in
the progeny of these plants (data not shown).
MERE1 Is Expressed during in Vitro Tissue Culture
As described above, MERE1 transposition was observed in Medicago plants produced using in vitro
tissue culture. To investigate the regulation of MERE1
activity under these conditions, its transcription activity was monitored in calli from 2HA and R108 lines cultivated on the SH3a callus-inducing medium (Cosson
et al., 2006). In the two experiments, MERE1 expression was transiently induced in both lines (Fig. 6A).
The induction in the 2HA line started 2 d after in vitro
culture and decreased after 7 d. For the R108 line, the
induction was delayed and reached a peak at 14 d. In
both lines, a decrease of the expression was observed
later on. In this northern-blot experiment, the probe
could not discriminate the different members of the
MERE1 family. Figure 6B shows that similar results
were obtained in reverse transcription (RT)-PCR experiments using oligonucleotides specific for the active MERE1-1 retroelement, suggesting that this copy
is active under the test conditions.
In order to better visualize the expression of the
complete MERE1 family present in the sequenced and
unsequenced parts of the genome, a PCR experiment
was done with nonspecific oligonucleotides that allow the amplification of a 471-bp region (positions
4,251–4,722 in MERE1-1) at the C-terminal extremity of
the GAG-POL protein. In this region, 43 positions
allow discrimination between the different MERE1
members. The PCR experiment was done using cDNA
of 14-d in vitro explants and 2HA genomic DNA
(gDNA), and 66 independent PCR fragments were
analyzed for cDNA and gDNA. The sequence analysis
showed that all MERE1 full-length members
were detected in gDNA (Supplemental Table S2). In
addition, incomplete copies present in the sequenced
genome as well as a limited number of unknown
MERE-like sequences, including some MERE1-1/
MERE1-4 and MERE1-5 closely related sequences,
Figure 6. Analysis of MERE1 expression in the Jemalong 2HA and R108
lines during in vitro culture. A, Northern-blot analysis of MERE1
expression. For each time point, 10 mg of total mRNA was loaded on
the gel and the blot was hybridized with the probe indicated in Figure
3A or with the Mtc27 constitutive gene probe. The 4.3-kb transcript
corresponds to a full-length MERE1 transcript. B, RT-PCR analysis of
MERE1-1 and MtPR1 expression. MERE1-1 expression was analyzed
using oligonucleotides specific for this element (MERE61-2/MERE61-4).
The EF1a gene was used as a constitutive gene control.
could also be detected. These latter are probably
present in the part of the genome not yet sequenced,
and for this reason we cannot know if they represent
complete MERE1 copies. Using the cDNAs, MERE1-3,
MERE1-4, and MERE1-5 sequences were not detected,
suggesting that these copies are not expressed under
these conditions. The other MERE1-related sequences
could be identified in the cDNA sample. Supplemental
Table S2 shows that the MERE1-1 element is the only
fully described element with a complete ORF and
identical 5# and 3# LTRs whose expression is induced
during the early stages of the tissue culture conditions.
This result is in agreement with its transposition
activity during tissue culture.
In order to test if, as described for other elements
(Casacuberta and Grandbastien, 1993; Grandbastien
et al., 1997; Vernhettes et al., 1997; Takeda et al., 1999),
MERE transcription was associated with stresses induced by the in vitro conditions, we studied the
expression of a pathogenesis-related (PR) protein
marker, MtPR1 (Szybiak-Strozycka et al., 1995), in the
experiment described above. Szybiak-Strozycka et al.
(1995) have shown that MtPR1 is most strongly similar
to pathogenesis-related proteins that are known to
be induced upon stress, pathogen attack, and abiotic
stimuli. PR up-regulation, therefore, is a good indication of various stress-related stimuli. MtPR1 expression was already detectable at the beginning of the in
vitro culture (T0) but increased 7 d after the beginning
of the experiment for 2HA and R108 lines (Fig. 6B).
This induction correlates with the MERE1 induction in
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A Mobile Retroelement in Medicago truncatula
these samples, in agreement with the fact that the in
vitro culture conditions (callus formation) used for
plant regeneration represent stress conditions. However, no specific induction of MERE1-1 expression
could be observed after culture of leaf explants cultivated in vitro on medium supplemented with aminoethoxyvinylglycine, 1-aminocyclopropane-1-carboxylic
acid, salicylic acid, or methyl jasmonate (data not
shown). This suggests that these stress-related hormones are not directly responsible for induction of
MERE1-1 expression in vitro. Similarly, 2,4-dichlorophenoxyacetic acid and benzylaminopurine growth
hormones used during in vitro regeneration were
unable to induce MERE1 expression on their own
(data not shown), suggesting that the expression of the
element is not directly controlled by these growth
hormones.
MERE1 Transcription Is Correlated with Its
Methylation Status
DNA methylation may regulate the transposition of
retroelements. For example, the transposition of Tto1
and Tnt1 in Arabidopsis (Hirochika et al., 2000; PerezHormaeche et al., 2008) or MAGGY in the fungus
Magnaporthe grisea (Nakayashiki et al., 2001) is controlled by methylation.
To examine the methylation status of the MERE1
element, the cytosine methylation pattern was determined for the MERE1 family using the bisulfite methylation profiling method (Reinders et al., 2008). With
this treatment, unmethylated cytosines are converted
to uracil, in contrast to 5-methylcytosine located
mainly in symmetrical CG and CNG (N = A or T) or
nonsymmetrical CHH (H = A, T, or C) sequences
(Frommer et al., 1992; Vanyushin, 2006). Bisulfitemediated cytosine conversion and subsequent PCR
create C-to-T transitions, which can be detected by
sequencing. In order to detect modification of the
MERE1 methylation status, gDNA was extracted from
2HA and R108 leaf explants cultivated on SH3a for 0,
4, 7, and 14 d of culture and treated with bisulfite (see
“Materials and Methods”). A 225-bp specific fragment
(positions 4,462–4,687; see “Materials and Methods”)
was selected to study the methylation status on the
MERE1 family (Supplemental Fig. S4A). This sequence
was chosen because it is the most accurate to detect
changes in the methylation pattern of the MERE
sequence as determined by the MethPrimer program
(Li and Dahiya, 2002). For each CG, CNG, and CHH
present in the sequence and for each time point of the
experiment, we determined the percentage of methylation (percentage of C unconverted to T; Fig. 7A;
Supplemental Fig. S4B). The results obtained for the
two lines (Jemalong and R108) are similar (Fig. 7A;
data not shown) and indicated that the MERE1 cytosines are highly methylated before the beginning of the
experiment and after 4 d of culture, when the expression of the element is still very low (Fig. 7). A significant
decrease of the methylation status can be detected after
7 d using the 2HA gDNA (14 d for R108), corresponding to the peak of induction of the element as shown
by northern blot. At 14 d (21 d for R108), the percentage
of mCG methylation increased again. This experiment
showed that the percentage of cytosine methylation of
this region is inversely correlated with the activity
of the retroelement, indicating a close connection between MERE1 expression and the methylation status of
the CG and CNG cytosines present in its sequence. The
CHH residues that were highly methylated at the
Figure 7. Methylation status at the MERE1 locus. The
graph represents the level of methylation (% methylation; y axis) at the different C residues in the
sequenced fragment. The DNA methylation level
was determined using the bisulfite sequencing
method and presented for each different sequence
motif: CG, CNG, and CHH (N = A or T; H = A, T, or
C). The position in the sequence indicates C residues.
For complete sequence analysis, see Supplemental
Fig. S4A. A, Percentage methylation of the CG and
CNG residues present within the analyzed sequence.
B, Percentage methylation of the CHH residues.
These residues are divided into two groups: those
with a methylation level superior to 20% at time 0,
and those with a methylation level inferior to 20%.
The results for representative CHH residues (CHH35,
CHH43, CHH57, CHH139, CHH151, and CHH154)
from the first group (.20%) and residues CHH112
and CHH145 from the second group (,20%) are
shown.
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Rakocevic et al.
beginning of the experiments also showed a transient
demethylation. However, this is not the case for the
residues that were much less methylated (Fig. 7B).
MERE1-1 Can Insert into Genes
In order to compare the nature of the sequences
flanking new MERE1-1 insertions and the ones of the
well-described Tnt1 tobacco element, 100 MERE1
flanking sequence tags (FSTs) and 288 Tnt1 Medicago
FSTs were analyzed. The flanking sequences were
classified in three classes: CDS (coding sequences),
repetitive (repetitive elements), and unknown when a
target sequence could not clearly be identified (Fig.
8A). Within the 100 MERE1-1 FSTs, we could identify
33 ESTs using BLASTn analysis (http://compbio.dfci.
harvard.edu/tgi/; E value of 10210). Similarly, a
BLASTx analysis (E value of 10210) showed that 33
sequences belong to the CDS class and nine to the
repetitive elements class. The same analysis using 288
Tnt1 FSTs shows nearly the same results: 28% of the
288 FSTs belong to the CDS class and 6% to the
repetitive elements. These results are in agreement
with those of Tadege et al. (2008), who showed using a
larger population that 34.1% of the Tnt1 inserts are in
ORFs, despite CDS representing only 15.9% of the M.
truncatula pseudogenome. This result indicates that
coding sequences are tagged at a similar frequency by
MERE1-1 and Tnt1. Moreover, the analysis of the
sequences flanking the two retroelements indicated
no clear consensus target site for the two elements (Fig.
8B). The GC composition of the 50 bp downstream of
the two elements, including the TSD sequence, is
roughly identical to the M. truncatula pseudogenome.
However, a small increase in the frequency of G/C
was observed at positions 1 and 5 of the TSD. In
addition, at position TSD+3, G and C frequency was
increased to 29% and 41%, respectively, instead of
17.5% each (Fig. 8B; Supplemental Table S3). Thus, as
for Tnt1, there is a modest preferences for GC versus
AT at several positions of the MERE1 integration site.
DISCUSSION
The nsp2-4 Mutant Phenotype Differs from Previously
Described nsp2 Mutants
Despite being an allele of nsp2-1 and nsp2-2, which
show root hair branching, nsp2-4, similar to the M.
truncatula nfp mutant (Amor et al., 2003), is characterized by an absence of root hair branching under our
test conditions. Different genetic background (R108
versus Jemalong) might explain the different root hair
responses. In line with these differences, we observed
that R108 wild-type plants showed reduced curling
and branching compared with the Jemalong wild-type
plants 2 d after infection, a time point when the
branching phenotype appears in the nsp mutants
(data not shown).
The NSP2 Gene Is Tagged by the Copia
MERE1 Retroelement
In this work, we have identified an active Medicago
retroelement (MERE1) that belongs to a family composed of five to 10 members in the M. truncatula
genome. Our study using M. truncatula ecotypes of
different geographical origin shows a constant MERE1
copy number. This suggests a relative stability of this
element copy number throughout the Medicago genus.
Figure 8. MERE1-1 has no target
sequence site specificity but tends
to insert within the ORF. A, Distribution of the genomic sequences
flanking newly transposed MERE1-1
(100 inserts) and Tnt1 (288 inserts). CDS are indicated in black,
repetitive sequences in gray, and
undetermined sequences in white.
B, Target site sequence analysis. A
total of 157 sequences were analyzed for the MERE1-1 element and
1,725 sequences for the Tnt1 element using the program WebLOGO
3.0. The size of the letters indicates
the frequency of each base pair. The
positions of the TSD are indicated
by the arrows.
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A Mobile Retroelement in Medicago truncatula
Three of the five copies, MERE1-1, -3, and -4, are
complete LTR retroelements, with complete GAG-POL
coding regions. Surprisingly, only one of these five
elements (MERE1-1) was active during tissue culture.
The two other members with intact coding capacity
were stable under our experimental conditions.
BLAST analysis and PCR experiments indicated that
MERE1-like elements are present in other legume
species like L. japonicus and Cajanus cajan, but we did
not detect related elements in Arabidopsis. Interestingly, the U5 region of the LTRs is highly conserved
between the elements in L. japonicus and M. truncatula,
whereas the U3 region, which contains the promoter
region, is more variable. This more variable sequence
of the promoter region may represent an evolutionary
adaptation to control the activity of the retroelement
(see below).
Interestingly, the sequenced part of the genome
contains only five solo LTRs, which have significant
sequence differences from the full-length MERE1
LTRs. This suggests that other MERE1-1-like elements
may exist in the nonsequenced part of the genome, but
it also reflects the low activity of the MERE1-1 family.
MERE1 Is an Active Retroelement in M. truncatula
The mutation induced by MERE1-1 was first detected in a M. truncatula R108 background. Our work
demonstrated that transposition occurred in 40% of
the R108 regenerated plants from a small mutant
collection, with an average new copy number of one
element per plant in the total collection, indicating a
mild activity of the element in this background during
tissue culture. The stability of the MERE1 retroelement
copy number in M. truncatula (and L. japonicus) might
result, as for Tnt1 (Grandbastien et al., 1994), from a
long coevolution of this particular family of retroelements with its host organism. Interestingly, this element was also found active in the M. truncatula
Jemalong 2HA line used to generate the Medicago
Tnt1 insertion mutant collection (www.eugrainlegume.
org). In this line, the MERE1 element was also activated
by the in vitro tissue culture procedure, used primarily
to activate the Tnt1 element. This experiment suggests
that the two retroelements might be activated under the
same tissue culture conditions and at the same time in
the mutant plants.
The analysis of the MERE1 integration site sequences showed that there is no hot spot for integration and no consensus sequence, except an increase of
G/C residue in the TSD+3 position, for MERE1 integration site. In addition, the analysis of 100 insertion
site sequences showed that, like Tnt1 (Tadege et al.,
2008), MERE1 inserts twice as much as expected inside
genes in M. truncatula.
The MERE1 Activity Is Detected during Tissue Culture
The induction of MERE1-1 during tissue culture
reaches its maximum at different time points depend-
ing on the genotype used (7 d for 2HA versus 14 d for
R108). A plausible explanation for these differences
comes from the genetic background and the regeneration history of both genotypes. R108 and 2HA were
subjected to several cycles of somatic embryogenesis
on different growth media to select highly regenerable
plants (Trinh et al., 1998; Rose et al., 1999). 2HA,
therefore, will have a different regeneration capacity
on a R108 medium, which was used herein to investigate MERE1 induction during tissue culture. Thus,
besides being genetically different, medium-dependent
callus growth may contribute to the observed shift in
MERE1-1 induction.
Plants with new MERE1-1 copies did not show any
transposition activity in their T2 generation, suggesting that the MERE1-1 activation was restricted to
tissue culture. The absence of transposition in further
generations suggests that this might be the consequence of an increase in methylation, thus silencing
and stabilizing the MERE1 retroelement in regenerated plants. Transposition activity in the course of
tissue culture could not be induced by stress-related or
plant growth hormones, since MERE1-1 expression
was not detected when either hormone was added
separately to the regeneration medium (see “Results”).
Our study indicated that only MERE1-1 transposes,
since no copies of the additional MERE1 could be
detected. The most likely explanation is that the remaining full-length MERE1 elements (MERE1-2, -4,
and -5) have lost their coding capacities because of the
incomplete ORF or aging restricts their transposition,
as they have sequence differences between the 3# and
5# LTRs. The identity between 5# and 3# LTR sequences
has been previously reported to be correlated to the
activity of retroelements in mouse (Ribet et al., 2004).
Those authors have shown that mobilization of MusD
transposon is correlated to 100% identity between its
LTRs, which is consistent with their recent transposition
and them being active. More recently, Maisonhaute
et al. (2007) have shown that the newly inserted 1,731
LTR retrotransposon copies in Drosophila show 100%
identity compared with older copies. An exception is
MERE1-3, which was not expressed (no cDNA detected) and was not recovered in any of the mutant
screens despite having an intact ORF and identical
LTRs. This suggests that MERE1-3 might be the target
of an epigenetic regulation. Its insertion in a particular
region of the M. truncatula genome might trigger
genomic position effects that could result from a single
phenomenon or a combination of diverse epigenetic
phenomena such as DNA hypermethylation, histone
modifications, or any other evolution of the chromatin
state. Epigenetic regulation of retrotransposons is a
known phenomenon reported in rice, Brassica rapa,
Drosophila, and many other organisms (Cheng et al.,
2006; Eickbush et al., 2008; Fujimoto et al., 2008). We
cannot know if the other copies detected in this analysis as cDNA and gDNA fragments represent MERE1
functional elements. Further investigations will shed
light on the underlying mechanisms responsible for
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Rakocevic et al.
the differential regulation of MERE1 copies in M.
truncatula.
We could not detect new copies of the Lotus MERE1like element in the progeny of transgenic Lotus plants
regenerated in vitro (data not shown). Interestingly,
transposition could also not be detected in Arabidopsis transgenic plants transformed with the R108
MERE1-1 element isolated from the MtNSP2 locus
(data not shown). It should be noted that both transgenic Arabidopsis plants and regenerated L. japonicus
plants were produced using transformation/regeneration methods not based on somatic embryogenesis.
This suggests that transposition of MERE1 is dependent on the genetic background used, on tissue culture
conditions, on the combination of both, or on other
unknown regulatory mechanisms. Such genotypeand tissue culture-dependent transposition activity
was described for Tnt1 in the M. truncatula 2HA line
(Iantcheva et al., 2009). It might thus be interesting
to regenerate MERE1-containing Arabidopsis or L.
japonicus plants using an embryogenesis-mediated
method in order to know if this procedure is crucial
for the activation of these elements.
Interestingly, a phylogenetic analysis including
most of the plant copia retroelement clades indicates
that several tissue culture-activated plant copia elements (MERE1-1, Tnt1, and Tto1) belong to the same
subfamily of the copia clade 1 (Supplemental Fig. S2),
suggesting that they may have retained common regulatory mechanisms that allow their activation during
tissue culture.
Transcription of MERE1 Correlates with
Partial Demethylation
Methylation is generally correlated with the repression of retroelements (Martienssen and Colot, 2001).
We observed a clear correlation between the expression of the element and a transient diminution of the
methylation status of the MERE1 element during the
early steps of our in vitro experiments. The transient
reduction of the methylation was particularly clear for
the CG residues and for one of the two methylated
CNG residues present in the analyzed sequence. The
highly methylated CHH residues also showed a transient demethylation. Thus, there is a clear correlation
between the methylation status of the element and its
transposition activity during in vitro culture. Such
tissue culture-induced demethylation was previously
reported in soybean (Glycine max) and maize (Zea
mays; Quemada et al., 1987; Kaeppler and Phillips,
1993). Although decrease in methylation is often associated with tissue culture, it is still unclear why this
happens. We can only hypothesize an adaptive response of the plant to tissue culture stress conditions
that allows a transcriptional activation of appropriate
genes. Reduced methylation of MERE1 suggests that
our in vitro culture conditions (somatic embryogenesismediated plant regeneration) might induce important
changes in the methylation status of the retroelement,
probably reflecting a transient demethylation of large
regions of the Medicago genome that might be responsible for the activation of the MERE1 retroelement. One
might speculate that this could also be the case for the
transposition of Tnt1. This is a likely scenario, since it
was shown in Arabidopsis that Tnt1 silencing is released in decrease in DNA methylation and polymerase IVa
mutants (Pérez-Hormaeche et al., 2008). Furthermore,
those authors report a negative correlation between
Tnt1 copy number and its silencing, which was shown
to be associated with short interfering RNAs, suggesting an RNA-directed DNA methylation.
MATERIALS AND METHODS
Plant Material
The NSP2-4 allele was originally called ms219. The first NSP2-1 allele was
described by Kalo et al. (2005). Medicago truncatula lines J5, 2HA (2HA3-9-103), and R108 (Trinh et al., 1998; Rose et al., 1999) were used as the wild type.
The M. truncatula accessions were provided by J.M. Prosperi (INRA UMR
DIAPC; http://www1.montpellier.inra.fr/BRC-MTR/) and were characterized previously using microsatellite markers as described by Ronfort et al.
(2006). DZA lines originate from Algeria, F and Salses lines from France, ESP
lines from Spain, GRC lines from Greece, and CRE lines from Crete. The
Jemalong Tnt1 insertion mutant collection was constructed in the frame of the
FP6 GLIP program (www.eugrainlegumes.org). GKB mutants were described
by Brocard et al. (2006). Plants for leaf explants were grown 4 weeks on SHb10
medium (EMBO Practical Course on the New Plant Model System Medicago
truncatula: Module 2; http://www.isv.cnrs-gif.fr/embo01/index.html) in environmentally controlled walk-in growth chambers at 24°C and 200 mmol
photon m22 s21 light intensity. MERE1-1 corresponds to the Mtr16 element
recently described by Wang and Liu (2008), localized on bacterial artificial
chromosome clone CR931743.
Bacterial Strains
The Escherichia coli strain XL1 Blue (Stratagene) was used for cloning and
propagation of different vectors. Agrobacterium tumefaciens EHA105 strain
(Hood et al., 1993) was used in all plant transformation experiments. Plant
transformation vectors were introduced into this strain by electroporation
(Mattanovich et al., 1989). For the nodulation tests, plants were inoculated 4 d
after germination using strain FSM.Ma (www.ccmm.ma) or Sinorhizobium
meliloti 1021 (NC 003047; Galibert et al., 2001) culture resuspended in sterile
water (optical density at 600 nm = 0.3).
Plant Tissue Culture
Regenerated plants and calli were obtained according to Cosson et al.
(2006). For time course experiments, plants were first cultivated in vitro
during 4 weeks, and leaf explants were scarified with surgical blades and
placed on SH3a medium as described by (Cosson et al., 2006) for 4 weeks in
two independent experiments.
Isolation of the NSP2-4 Allele
The nsp2-4 allele was identified by forward genetic screening of an R108derived T-DNA mutant collection (Brocard et al., 2006) inoculated with
Sm1021. The nodulation test was carried out in the greenhouse with approximately 30 T2 plants grown in a sand:perlite mix (1:3, v/v) for 4 weeks after
inoculation. Nonnodulating plants were screened a second time 3 weeks later
after a second inoculation. Nonnodulating plants recovered from the second
screen were grown to seed in standard soil in greenhouse conditions.
Root Hair Observations
Seeds were germinated in sterile water overnight at room temperature.
Plants were cultivated on buffered nodulation medium (Ehrhardt et al., 1992)
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A Mobile Retroelement in Medicago truncatula
for 2 d and inoculated with a suspension of bacteria at optical density at 600
nm = 0.3. Plants were stained for 15 min with 0.02% methylene blue, rinsed
three times with liquid buffered nodulation medium, and observed with a
Polyvart Reichert microscope equipped with a Nikon DXM1200 camera.
control amplifications. Twenty-five cycles of PCR (94°C for 30 s, 56°C for 30
s, and 72°C for 30 s) were carried out for MtEF1a and 30 cycles for other genes.
Amplification products were analyzed using 1.5% agarose gel electrophoresis.
Segregation Analysis
Mapping Population
Two independent T1 plants of ms219 were backcrossed with the J5
accession. Six independent crosses were obtained, and the F2 population
was tested for its symbiotic phenotype as described above. The mapping
population contained 45 nod2 plants and 113 nod+ plants. gDNA was
extracted from 137 plants (41 nod2 plants and 96 nod+ plants) and used for
the PCR mapping experiments. Genetic markers are listed in Supplemental
Table S1.
Southern-Blot Analysis
Ten micrograms of DNA was digested with restriction enzyme under
conditions specified by the suppliers and separated on a 13 Tris-acetate
EDTA, 1% agarose gel overnight at 1 V cm21. Southern blotting was performed
as described by Sambrook et al. (1989), and probe hybridization as described
by Church and Gilbert (1984). The MERE1 probe consists of a 520-bp PCR
fragment located between MERE1 nucleotides 4,198 and 4,718 (Fig. 3A).
Transposon Display Analysis
MERE1 borders were isolated using the transposon display method.
gDNA was extracted, and 1 mg was digested with AseI restriction enzyme
for 3 h. The enzyme was inactivated at 65°C for 20 min. AseI adaptors (Aseadap1 [5#-CCCCTCGTAGACTGCGTACC-3#] plus Ase-adap2 [5#-TAGGTACGCAGTCTACGA-3#]) were ligated (T4 DNA ligase; Fermentas) at 16°C
overnight. Preamplification (PCR1) was done using MERE2 or MERE51/
MERE52 and Ase1 primers, and the PCR products were diluted 1:100 for a
second PCR (PCR2) using MERE1, MERE61, MERE62, MERE63 or MERE64,
and Ase2 primers.
The PCR1 program was as follows: 94°C for 2 min; five times 94°C for 30 s,
60°C for 20 s, and 72°C for 1.5 min; five times 94°C for 30 s, 58°C for 20 s, and
72°C for 1.5 min; 20 times 94°C for 30 s, 56°C for 20 s, and 72°C for 1.5 min. The
PCR2 program was as follows: 94°C for 2 min; 10 times 94°C for 30 s, 55°C for
20 s, and 72°C for 1.5 min; 25 times 94°C for 20 s, 52°C for 20 s, and 72°C for 1.5.
PCR products were analyzed on 1.5% agarose gels.
Primers were as follows: MERE1 (5#-AGCCCTTTTGTCAAACATGTATTA-3#), MERE2 (5#-AATGTTGGCACCGAAAATCTGAATGGT-3#), MERE61
(5#-AACAAAAGGTGACTTTATGGCTT-3#), MERE62 (5#-CAACAAAATGTGGCTTTACTTGT-3#), MERE63 (5#-CAACAAAAGTGGCTTTACCACT-3#),
MERE64 (5#-CAACATGATTTGACTTGGCACA-3#), MERE51 (5#-GCCTCAACCATTTTCATTTATTACCAT-3#), and MERE52 (5#-TGCCTCAACCAMTYSMATTTAWGGCA-3#).
Northern Blot
Total RNA was extracted from 2 g of plant material according to Kay et al.
(1987). The RNA concentration was measured with a spectrophotometer (ND100; Nanodrop). Ten micrograms of RNA was used for northern blotting
according to Sambrook et al. (1989). The MERE1 probe is the same as the one
used for Southern blotting. Constitutively expressed gene Mtc27 was used as a
control (TC 85211; Verdier et al., 2008).
RT-PCR
For the segregation analysis, we used primers NSP2-5# (5#-CCGGCAGGCGATTAACCTCCTT-3#), NSP2-3# (5#-GCATCCTATAAATCAGAATCTGAA-3#), and MERE1 (5#-AGCCCTTTTGTCAAACATGTATTA-3#). Thirty
cycles of PCR were carried out at 94°C for 30 s, 56°C for 30 s, and 72°C for 45
s. Amplification products were analyzed using 0.7% agarose gel electrophoresis.
DNA Methylation Analysis
gDNA was extracted from 2HA leaf explants after 0, 4, 7, and 14 d of in
vitro culture on SH3a medium. Two micrograms of each DNA sample was
subjected to bisulfite treatment using the EpiTect Bisulfite Modification Kit
(Qiagen), which includes a DNA protective buffer.
Primers were designed using the MethPrimer Web site (http://www.
urogene.org/methprimer/index.html).
The Bi1 (5#-TAACAACTAAAATTAACAAAATAAAAAA-3#) and Bi2
(5#-GAATGAAAAGTTGGATTTTTTGTAA-3#) primers amplified the converted (+)DNA strands corresponding to MERE1 positions from 4,462 to
4,687 bp. Three aliquots of 2 mL of converted DNA were prepared for each
time point. In addition to the DNA samples, controls of gDNA without
bisulfite conversion and template-free controls (“no-template control”) were
prepared in parallel. Amplification could not be detected on nonconverted
DNA using Bi1 and Bi2 primers. Primers BiWT1 (5#-TAGCAACTGGAGTTGGCAAGGTAGAGAG-3#) and BiWT2 (5#-GAATGAAAAGCTGGATTCCTTGCAA-3#) were designed to amplify the nonconverted sequences. Using
these primers, we obtained PCR products on nonconverted DNA but no
products on bisulfite-converted DNA. PCR (94°C for 2 min, 50°C for 30 s, and
72°C for 30 s, 35 cycles) was performed according to the supplier with 0.4 units
of Taq polymerase (Eurobio). PCR products for each time point were pooled
and cloned in the pGEM-T Easy Vector (Promega). For each time point,
between 18 and 21 sequences were analyzed between base pairs 4,462 and
4,687 of the MERE1 sequence.
Bioinformatics Analysis
Sequences were analyzed by BLAST (www.ncbi.nlm.nih.gov/BLAST/),
BioEdit Sequence Alignment Editor (1997–2007; Tom Hall), and Multalin
(http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.html). The alignments of the GAG-POL regions were conducted using ClustalX (Thompson
et al., 1997). Relationship tree building was conducted using MEGA version 4
(Tamura et al., 2007).
FST sequences were generated in the framework of the EU-GLIP program
by the Genoscope (www.genoscope.cns.fr) and analyzed using the program
WebLOGO 3.0 (http://weblogo.threeplusone.com/).
Sequence data from this article can be found in the GenBank/EMBL
data libraries under accession numbers FI495319 to FI495418 for the FSTs.
Other accession numbers are as follows: MERE1-1-J5, FJ544851; MERE1-2-J5,
FJ544852; MERE1-3-J5, FJ544853; MERE1-4-J5, FJ544854; MERE1-5-J5,
FJ544855; MERE1-1-R108-1, FJ544856; MERE1-LIKE-1, FJ544857; and
MERE1-LIKE-2, FJ544858. Tnt1 FSTs sequences were deposited at http://
bioinfo4.noble.org/mutant/.
Supplemental Data
Samples were collected at 0, 2, 7, 14, and 28 d of in vitro culture and
immediately frozen in liquid nitrogen. Total RNA for RT-PCR analysis was
prepared from M. truncatula samples using the RNeasy Mini Kit (Qiagen).
Residual gDNA was removed using RNase-free DNase (Qiagen). From 2 mg of
total RNA, cDNA was generated using SuperScript reverse transcriptase
(GibcoBRL/Life Technologies). The MERE1-specific primer pair used was
MERE61-2 (5#-GTGAACATGTGTGAACACATAAGCC-3#) and MERE61-4
(5#-GCAAGGGACATGCTATTTATAGGACC-3#). PRL-1-specific primers were
designed against the PRL-1 gene (X79778). The constitutively expressed M.
truncatula elongation factor gene (MtEF1a; EF1a [5#-AGTCTCTCTCTGCGGCTGAG-3#] and EF1b [5#-CGATTTCATCGTACCTAGCCTT-3#]) was used in
The following materials are available in the online version of this article.
Supplemental Figure S1. RT-PCR analysis of nodulin gene expression in
the wild type and ms219 after infection by S. meliloti.
Supplemental Figure S2. Evolutionary relationships of MERE1 LTRs and
related solo LTRs.
Supplemental Figure S3. Evolutionary relationships of 155 copia-related
retroelements.
Supplemental Figure S4. Methylation status at the MERE1 locus.
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Rakocevic et al.
Supplemental Table S1. Sequences of genetic markers on linkage groups
1, 3, and 5.
Supplemental Table S2. MERE1-1 is the only member of the MERE1
family with all requirements for transposition.
Supplemental Table S3. Base composition of MERE1-1 insertion sites.
ACKNOWLEDGMENTS
We are very grateful to Valérie Geffroy and Gabriella Endre for kind help
in the marker design and analysis of results. We are very grateful to Lene
Heegaard Madsen and Jens Stougaard, who provided us with gDNA of the
progeny of transgenic L. japonicus plants. Thanks to Peter Kalo and Giles
Oldroyd, who provided us with the nsp2-1 mutant. Thanks to Pavel
Neumann and Jiri Macas for the sequence data of the plant copia elements.
We are grateful to Drs. M. Schultze, R. Benlloch, and M.A. Grandbastien for
careful reading of the manuscript.
Received March 3, 2009; accepted July 29, 2009; published August 5, 2009.
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