mere1, a low-copy-number copia-type retroelement...mere1, a low-copy-number copia-type retroelement...

MERE1, a Low-Copy-Number Copia-Type Retroelementin Medicago truncatula Active during Tissue Culture1[C][W]

Alexandra Rakocevic, Samuel Mondy, Leıla Tirichine, Viviane Cosson, Lysiane Brocard, Anelia Iantcheva,Anne Cayrel, Benjamin Devier, Ghada Ahmed Abu El-Heba2, and Pascal Ratet*

Institut des Sciences du Vegetal, 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 aninsertion 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 thelong terminal repeats of these different copies. This retroelement family is present in all M. truncatula ecotypes tested but alsoin other legume species like Lotus japonicus. It is active only during tissue culture in both R108 and Jemalong Medicagoaccessions and inserts preferentially in genes.

Transposable elements are mobile genetic elementspresent in a wide range of organisms from bacteria toeukaryotes and are usually classified in two differentgroups. Class I elements, called retrotransposons,transpose through an RNA intermediate that is reversetranscribed into a linear double-stranded DNA beforeits integration into the host genome (copy-and-pastemechanism). Class II elements, called DNA trans-posons, transpose directly via a DNA intermediate(cut-and-paste mechanism). The replicative mode oftransposition of class I retrotransposons may rapidlyincrease their copy number, which can be extremelyhigh in eukaryote genomes.

Because they can invade genomes, minimizing theircopy number is particularly important to maintain thegenome integrity, and active retroelements are gener-ally found in low-copy-number families (Madsenet al., 2005). These elements are present in plants andmammals, but long terminal repeat (LTR) transposonsappear to be rare in the latter. LINEs, SINEs, Ty3-

gypsy, and Ty1-copia appear to be abundant in plants(Kumar and Bennetzen, 1999). Transposable elementsare usually located in intergenic regions and tend toaccumulate in centromeres, telomeres, and heterochro-matic regions. An analysis of 233 Mb of the Medicagotruncatula genome shows that retroelements constituteabout 9.6% of the currently available genomic se-quence, and the majority of the LTR retroelementsbelong to either the copia or gypsy superfamily (Wangand Liu, 2008).

Despite the fact that they are widespread andabundant, only a few of them have been described asactive in plants: Tnt1 (Grandbastien et al., 1989), Tto1(Hirochika et al., 2000), Tos17 (Hirochika, 1997), andLORE1 and LORE2 (Fukai et al., 2008). New transpo-sition events in genomes have been noticed, but thecorrelation between transcription induction and trans-position is not always obvious. Plant retroelements canbe activated by tissue culture in tobacco (Nicotianatabacum [Tto1]; Hirochika, 1993), rice (Oryza sativa[Tos17]; Hirochika et al., 1996; Miyao et al., 2003), andM. truncatula (Tnt1; d’Erfurth et al., 2003). Plant retro-elements are also activated by wounding or pathogenattack (Mhiri et al., 1997; Takeda et al., 1999; Melayahet al., 2001). For other plant mobile retroelements, theactivation conditions have not been established(LORE1; Madsen et al., 2005). The main characteristicof these retroelements is their ability to insert ran-domly in the genome and to alter gene functionfollowing disruption. The creation of this genetic var-iability might play an important role as a source ofgenome plasticity needed for plant evolution (Wessleret al., 1995).

Retroelements carry in their LTR region, which canbe subdivided into U3, R, and U5 regions, cis-actingsequences that control their expression in the host(Kumar and Bennetzen, 1999). Specific sequences inthe LTR U3 regions have been shown to be involved in

1 This work was supported by a fellowship from the FrenchMinistere de l’Education Nationale, de l’Enseignement Superieur etde 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 aUNESCO-L’OREAL-cosponsored Fellowship for Young Women inLife Science-2006 to G.A.A.E.-H.

2 Present address: Agricultural Genetic Engineering ResearchInstitute, Agricultural Research Centre, 9 Gamaa St., Giza 12619,Egypt.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed 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 inblack 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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the induction of the retroelement transcription byvarious biotic and abiotic stress factors (Casacubertaand Grandbastien, 1993; Vernhettes et al., 1997; Takedaet al., 1999). The variability observed in these U3regions among the different subfamilies of Tnt1 isthought to be associated with the ability of thesepromoters to respond to different signaling stressmolecules (Vernhettes et al., 1997; Araujo et al., 2001;Beguiristain et al., 2001). DNA methylation seems tobe an important factor controlling retroelement ex-pression in plants (Martienssen and Colot, 2001), be-cause high levels of cytosine methylation have beenassociated with transcriptional inactivity. This controlprocess is apparently different between mammals andplants. Repetitive elements are methylated in bothorganisms, but whereas most mammalian exons aremethylated, plant exons are generally not. Thus, tar-geting methylation specifically to transposons appearsto be restricted to plants (Rabinowicz et al., 2003).Transcription reduction of Tto1, Ttnt1, and Tos17 wascorrelated to their methylation status in Arabidopsis(Arabidopsis thaliana) and rice (Hirochika et al., 2000;Liu et al., 2004; Perez-Hormaeche et al., 2008), but thefactors controlling this methylation are not clearlyunderstood. Moreover, posttranscriptional regulationof the transposition is also an important step of theretrotransposition. In Neurospora crassa, the LINE1-likeretroelement is repressed by the posttranscriptionalgene silencing machinery independently of DNAmethylation (Nolan et al., 2005). In contrast, theDNA methylation machinery had no obvious effectson its expression. In Saccharomyces cerevisiae, Ty1 copynumber control occurs by both transcriptional andposttranscriptional cosuppression (Garfinkel et al.,2003).We describe hereMedicago RetroElement1-1 (MERE1-

1), an active retroelement in M. truncatula. This retro-element belongs to a small copia-like retroelementfamily of five to 10 members corresponding to theMtr16 family defined by Wang and Liu (2008). Thisfamily is also present in other legumes. MERE1-1 hastransposed in severalM. truncatulamutant collections,and its transposition activity is correlated to in vitrotissue culture and to methylation of its sequence.

RESULTS

Identification of MERE1, an Active Retroelementin M. truncatula

During a screen for M. truncatula nodulation mu-tants in a T-DNA mutant collection (Scholte et al.,2002), a nonnodulating (nod2) mutant (ms219) wasidentified (Brocard et al., 2006). We have shown thatthe corresponding (somaclonal) mutation does notcorrelate with the presence of the T-DNA in thismutant line. No root hair deformation or primordiumformation was observed after inoculation of the mu-tant with Sinorhizobium meliloti, indicating that thesymbiotic process was blocked early (Fig. 1), similarly

to the nfpmutant (Amor et al., 2003). The expression ofmarker 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 Nodfactor perception (Supplemental Fig. S1). Allelic testsperformed between ms219 and other mutants affectedin early Nod factor signaling, nfp (Amor et al., 2003),

Figure 1. Phenotype of the nsp2 nonnodulating mutant. A and B, Wild-type (A) and nsp2-4 (B) plants inoculated with S. meliloti FSM.Mastrain. C, Light microscopy of wild-type noninoculated root hairs. Dand E, Curled wild-type root hairs or forming “shepherd’s crook”observed after inoculation with Rm1021 strain. F, Nonnoculated roothair in MtNSP2. G, Noncurling MtNSP2 root hair inoculated withRm1021 strain. [See online article for color version of this figure.]

A Mobile Retroelement in Medicago truncatula

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dmi1, dmi2, and dmi3 (Catoira et al., 2000), indicatedthat these genes were not affected in ms219 (data notshown). Because the phenotype and the allelic testsindicated that the mutation could correspond to a newsymbiotic locus, the mutation was mapped usingcrosses between the ms219 mutant (backgroundR108) and the Jemalong 5 (J5) line. The F2 populationsfrom six independent crosses were used for mappingusing markers based on PCR fragment-length poly-morphisms (see “Materials and Methods”; Sup-plemental Table S1). The mutation was located to a3-centimorgan region on chromosome 3 borderedby bacterial artificial chromosomes AC149303 andAC126782 (www.medicago.org). This genomic regionincludes the NSP2 gene (Kalo et al., 2005). Therefore,this gene was considered as a candidate for the sym-biotic gene despite the different phenotype describedfor the known nsp2 mutant (root hair branching).Amplification of the NSP2 coding sequence in threeindependent ms219 plants using long-range DNApolymerase demonstrated that the NSP2 sequencecontained an insert of approximately 5.3 kb, whichwas likely responsible for the mutation (data notshown). Sequencing of the insert identified a retro-transposon, inserted at base pair 1,062 (Fig. 2A) of theNSP2 coding sequence. We named this transposonMERE1-1.

To demonstrate that the insertion of MERE1-1 intothe NSP2 gene cosegregated with the mutant pheno-type, the wild type and the nsp2 mutant allele, namednsp2-4 hereafter, were PCR amplified using primersspecific for both alleles (Fig. 2B). This showed that thensp2-4::MERE1-1 insertion cosegregated with the nod2

phenotype. Finally, to prove that the insertion in the

NSP2 gene is responsible for the phenotype, the nsp2-4allele 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 ofCopia Retroelements

The MERE1-1 retroelement identified at the NSP2locus is a 5,300-bp-long element and has two longterminal repeat (LTR) regions of 574 bp (Fig. 3A). Thiselement is flanked by a 5-bp duplication of host DNAcorresponding to the target site duplication (TSD). Asingle 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 similaritywith the GAG-POL protein of plant retroviral ele-ments. The location of the reverse transcriptase do-main behind the endonuclease (endo) classifiesMERE1 as a Ty1-copia retrotransposon.

BLAST searches against the M. truncatula Jemalonggenome sequence allowed the identification of fivecomplete MERE1 elements, numbered MERE1-1 toMERE1-5, corresponding to the Mtr16 family recentlydescribed by Wang and Liu (2008; for accession num-bers, see “Materials and Methods”). MERE1-1 hasbeen identified as the element present at the NSP2locus. Three of the MERE1 elements have the 1,305-amino acid full-length ORF (MERE1-1, MERE1-3, andMERE1-4). TheMERE1-2 sequence has an additional Tat nucleotide 2,852, creating a frame shift generating atruncated 707-amino acid ORF. TheMERE1-5 sequencecontains a stop codon at position 1,950, resulting in atruncated 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 (graybox) and variable N-terminal region (black box) are indicated. Positions are given in base pairs relative to the AUG.MERE1-1 isinserted at base pair 1,062 in the variable N-terminal region (3#–5# direction). nsp2-1 and nsp2-2 are two fast-neutron-generatedmutants containing a 435-bp in-frame deletion. nsp2-3 is an ethylmethane sulfonate mutant allele. B, Cosegregation of theMERE1 insertion in theNSP2 locus (NSP2-4::MERE1) with the nonnodulating phenotype of nsp2-4. A total of 41 F2 nod2 and 96F2 nod+ plants from a backcross between R108 NSP2-4 and J5 NSP2 were used to PCR amplify wild-type or mutant loci (theresults for 10 nod2 and 10 nod+ plants are presented). The absence of amplification products usingNSP2-specific primers (NSP2-5#/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 theMERE1 insertion. Thenod+ individuals are either wild type (no NSP2-MERE1 product) or heterozygous (presence of the two PCR products) for theNSP2-4::MERE1 insertion.

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Figure 3. Structure of theMERE1-1 retroelement representing theMERE1 family. A, Schematic representation ofMERE1-1. Grayboxes at the two extremities represent LTRs, divided into three parts: U3 (promoter), R (polyadenylation signal), and U5. Shadedboxes indicate regions showing homology with characteristic retrotransposon proteins. The hatched box below the RT regionrepresents the DNA fragment used as a probe in Southern- and northern-blot analyses. From N to C terminus, the uniqueMERE1-1ORF shows extensive amino acid homology to copia GAG (structural core proteins), PROT (protease involved in maturation ofGAG 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 shownbelow, as well as the complementary region between PBS and the 3# end of legume tRNAmet. B, Sequence comparison of the LTRregions from MERE1-1 to MERE1-5. The U5 region shows a high level of identity between the five sequences, whereas the U3region shows significant variability. The putative TATA box is indicated by the box. The positions of the different oligonucleotidesused in this study are indicated by arrows above the sequences. MERE6N and MERE5N represent the positions of the specificoligonucleotides MERE61, MERE62, MERE63, MERE64, MERE51, and MERE52 (for sequences, see “Materials and Methods”).[See online article for color version of this figure.]





ORF elements (MERE1-1, -3, and -4) could representactive retroelements, and the two others could repre-sent inactive ones. Comparison of the MERE1 familyLTR sequences shows conservation of the U5 regionbut 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. BLASTnsearch analysis indicated the presence of at least fivesolo LTRs related to theMERE1 family in theMedicagopseudogenome. Phylogenetical analysis indicated thatnone of these correspond to the LTRs of the MERE1-1to MERE1-5 elements (Supplemental Fig. S2).

The MERE1 Element Is Present at Low Copy Number inM. truncatula and Other Legume Species

To evaluate the copy number of the MERE1 elementin the entire Medicago genome, a Southern-blot analy-sis was performed using a probe corresponding to the3# extremity of the polymerase region (Fig. 3A). Thisanalysis indicates that the MERE1 family is present atmoderate copy number in the different lines analyzed(Fig. 4A), suggesting that only a few additional copiesare present in the heterochromatic unsequenced re-

gion of the genome. To analyze the MERE1 familymembers in more detail, a retrotransposon displaymethod (see “Materials and Methods”) was used,which allowed a better discrimination of the differentMERE1 elements present in the genome. Figure 4Bshows that many bands are common to J5 and therelated 2HA line and to R108 and its derivative nsp2-4.However, a polymorphism is detected between theJemalong and R108 lines. Similarly, using this tech-nique, a polymorphism can be detected between thedifferent lines from a small M. truncatula ecotypecollection (Fig. 4C), despite a rather constant copynumber of the element in these different lines. Bandsshared by some of these ecotypes (e.g. F83.005.9,DZA0517, F20061A, F34024A, and ESP158A) wereidentified, probably indicating a common geographi-cal origin.

Data mining also indicated thatMERE1-1 belongs toclade 1 (Wang and Liu, 2008) of copia plant retroele-ments and that MERE1-like retroelements are presentin different legume species (Supplemental Fig. S3).The MERE1 family is related to the Cajanus Panzeeelement described by Lall et al. (2002), and we couldidentify at least two full-length retroelements withLTR sequences similar to MERE1 in the Lotus japonicusMG.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 isindicated in Figure 3A. B, Transposon display of MERE1 5# insertion sites in wild-type and NSP2-4 mutant lines. The fragmentslabeled with asterisks represent copies of theMERE1-1 element different between 2HA and J5 lines. DNAwas digested with theAseI 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]) wasused as a negative control for this experiment. The average number of retroelements in the different ecotypes is less than 10. Theorigin of the different Medicago lines is given in “Materials and Methods.” DNAwas digested with the AseI restriction enzyme.PCR amplification was done using the oligonucleotide pairs MERE2/Ase1 and MERE1/Ase2.

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sequences from these two elements are 65% similar,and only one L. japonicus element has a completeGAG-POL protein. The analysis also indicates thatclosely related elements are present in Populus tremulusand Ipomoea batatas plants.

MERE1 Transposes in in Vitro-Produced

M. truncatula Plants

Because the MERE1 copy number is low and seemsto be stable in wild-type Medicago plants, we hypoth-esized that transposition at the NSP2 locus was in-duced by the tissue culture treatment in our Medicagotransgenic plants. Thus, in order to rule out that thetransposition of the MERE1 element was restricted tothe ms219 nod2 mutant, the presence of MERE1 addi-tional copies was monitored in transgenic plants ofdifferent mutant collections generated by in vitro cul-ture in our laboratory.

MERE1 transposition was first tested using twoMedicago R108 collections: the Tnt1 R108 collection(Tnk mutants, 250 lines; d’Erfurth et al., 2003) and theT-DNA collection (GKB mutants, 500 lines; Scholteet al., 2002; Brocard et al., 2006). The different MERE1borders produced in these experiments suggest thattransposition occurred in some of the mutant lines(Fig. 5, A and B), because the pattern of fragmentsobserved for these plants is unique. Altogether, thisexperiment demonstrates that transposition occurredin 73 plants over a total of 183 analyzed plants (40% ofplants with new transposition events). This representsone to more than 10 new insertions in the regeneratedplants and an average of one new copy per plant inthese collections.

MERE1 activity was also tested in regeneratedJemalong 2HA plants (Tnt1 insertion mutant collec-tion). As the LTRs of the different MERE1 memberspresent in the Jemalong genome have differences in

Figure 5. Transposon display ofMERE1 elements in in vitro-regen-erated plants. DNA was digestedwith the AseI restriction enzyme.The additional fragments detectedin lines marked with asterisks cor-respond to new insertion sites ofMERE1. Bands larger than 1,500 bpare PCR experimental artifacts. A,Tnk23 regenerated plants fromthe Tnt1 collection (R108 back-ground); five out of 23 analyzedplants are shown. PCR amplifica-tion was done using the oligonu-cleotide pairs MERE51+MERE52/Ase1 and MERE61/Ase2. B, GKBregenerated plants from the T-DNAcollection (R108 background); 16out of 39 analyzed plants areshown. PCR amplification wasdone using the oligonucleotidepairs MERE51+MERE52/Ase1 andMERE61/Ase2. C and D, Transposi-tion of the variousMERE1 elementsin regenerated 2HA Jemalongplants. The analysis of eight out of96 lines is shown. PCR amplifica-tion was done using the oligonucle-otide pairs MERE51+MERE52/Ase1for PCR1 followed byMERE61/Ase2for MERE1-1, MERE62/Ase2 forMERE1-2/MERE1-3, MERE63/Ase2for MERE1-4, and MERE64/Ase2for MERE1-5 for PCR2.





the 3# extremity, specific primers were designed toallow the specific amplification of the different trans-poson borders (MERE1-2 and MERE1-3 cannot bediscriminated by this technique). This experiment(Fig. 5, C and D) shows that new MERE1-specificfragments could be detected in some of the transgenicplants, indicating that transposition of MERE1 occursin a subset of the regenerated plants from this collec-tion. Surprisingly, we were able to detect the presenceof new copies only for the MERE1-1 element, indicat-ing that it could be the only active/mobile retroele-ment during tissue culture in Medicago.

PCR amplification of the MERE1-specific borders ina larger number of regenerated Jemalong 2HA andR108 plants confirmed that MERE1-1 is indeed theonly active retroelement in these two plants (data notshown). In addition, this analysis indicates that in thetwo Medicago lines transposition is restricted to tissueculture, because no new copies could be detected inthe progeny of these plants (data not shown).

MERE1 Is Expressed during in Vitro Tissue Culture

As described above, MERE1 transposition was ob-served in Medicago plants produced using in vitrotissue culture. To investigate the regulation of MERE1activity under these conditions, its transcription activ-itywasmonitored in calli from 2HAandR108 lines cul-tivated on the SH3a callus-inducing medium (Cossonet al., 2006). In the two experiments, MERE1 expres-sion was transiently induced in both lines (Fig. 6A).The induction in the 2HA line started 2 d after in vitroculture and decreased after 7 d. For the R108 line, theinduction was delayed and reached a peak at 14 d. Inboth lines, a decrease of the expression was observedlater on. In this northern-blot experiment, the probecould not discriminate the different members of theMERE1 family. Figure 6B shows that similar resultswere obtained in reverse transcription (RT)-PCR ex-periments using oligonucleotides specific for the ac-tive MERE1-1 retroelement, suggesting that this copyis active under the test conditions.

In order to better visualize the expression of thecomplete MERE1 family present in the sequenced andunsequenced parts of the genome, a PCR experimentwas done with nonspecific oligonucleotides that al-low the amplification of a 471-bp region (positions4,251–4,722 inMERE1-1) at the C-terminal extremity ofthe GAG-POL protein. In this region, 43 positionsallow discrimination between the different MERE1members. The PCR experiment was done using cDNAof 14-d in vitro explants and 2HA genomic DNA(gDNA), and 66 independent PCR fragments wereanalyzed for cDNA and gDNA. The sequence analysisshowed that all MERE1 full-length memberswere detected in gDNA (Supplemental Table S2). Inaddition, incomplete copies present in the sequencedgenome as well as a limited number of unknownMERE-like sequences, including some MERE1-1/MERE1-4 and MERE1-5 closely related sequences,

could also be detected. These latter are probablypresent in the part of the genome not yet sequenced,and for this reason we cannot know if they representcomplete MERE1 copies. Using the cDNAs, MERE1-3,MERE1-4, and MERE1-5 sequences were not detected,suggesting that these copies are not expressed underthese conditions. The other MERE1-related sequencescould be identified in the cDNA sample. SupplementalTable S2 shows that the MERE1-1 element is the onlyfully described element with a complete ORF andidentical 5# and 3# LTRs whose expression is inducedduring the early stages of the tissue culture conditions.This result is in agreement with its transpositionactivity during tissue culture.

In order to test if, as described for other elements(Casacuberta and Grandbastien, 1993; Grandbastienet al., 1997; Vernhettes et al., 1997; Takeda et al., 1999),MERE transcription was associated with stresses in-duced by the in vitro conditions, we studied theexpression of a pathogenesis-related (PR) proteinmarker, MtPR1 (Szybiak-Strozycka et al., 1995), in theexperiment described above. Szybiak-Strozycka et al.(1995) have shown thatMtPR1 is most strongly similarto pathogenesis-related proteins that are known tobe induced upon stress, pathogen attack, and abioticstimuli. PR up-regulation, therefore, is a good indica-tion of various stress-related stimuli. MtPR1 expres-sion was already detectable at the beginning of the invitro culture (T0) but increased 7 d after the beginningof the experiment for 2HA and R108 lines (Fig. 6B).This induction correlates with theMERE1 induction in

Figure 6. Analysis ofMERE1 expression in the Jemalong 2HA and R108lines during in vitro culture. A, Northern-blot analysis of MERE1expression. For each time point, 10 mg of total mRNA was loaded onthe gel and the blot was hybridized with the probe indicated in Figure3A or with the Mtc27 constitutive gene probe. The 4.3-kb transcriptcorresponds to a full-length MERE1 transcript. B, RT-PCR analysis ofMERE1-1 and MtPR1 expression. MERE1-1 expression was analyzedusing oligonucleotides specific for this element (MERE61-2/MERE61-4).The EF1a gene was used as a constitutive gene control.

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these samples, in agreement with the fact that the invitro culture conditions (callus formation) used forplant regeneration represent stress conditions. How-ever, no specific induction of MERE1-1 expressioncould be observed after culture of leaf explants culti-vated in vitro on medium supplemented with amino-ethoxyvinylglycine, 1-aminocyclopropane-1-carboxylicacid, salicylic acid, or methyl jasmonate (data notshown). This suggests that these stress-related hor-mones are not directly responsible for induction ofMERE1-1 expression in vitro. Similarly, 2,4-dichloro-phenoxyacetic acid and benzylaminopurine growthhormones used during in vitro regeneration wereunable to induce MERE1 expression on their own(data not shown), suggesting that the expression of theelement is not directly controlled by these growthhormones.

MERE1 Transcription Is Correlated with ItsMethylation Status

DNA methylation may regulate the transposition ofretroelements. For example, the transposition of Tto1and Tnt1 in Arabidopsis (Hirochika et al., 2000; Perez-Hormaeche et al., 2008) or MAGGY in the fungusMagnaporthe grisea (Nakayashiki et al., 2001) is con-trolled by methylation.To examine the methylation status of the MERE1

element, the cytosine methylation pattern was deter-mined for the MERE1 family using the bisulfite meth-ylation profiling method (Reinders et al., 2008). Withthis treatment, unmethylated cytosines are convertedto uracil, in contrast to 5-methylcytosine locatedmainly in symmetrical CG and CNG (N = A or T) ornonsymmetrical CHH (H = A, T, or C) sequences

(Frommer et al., 1992; Vanyushin, 2006). Bisulfite-mediated cytosine conversion and subsequent PCRcreate C-to-T transitions, which can be detected bysequencing. In order to detect modification of theMERE1 methylation status, gDNAwas extracted from2HA 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 theMERE1 family (Supplemental Fig. S4A). This sequencewas chosen because it is the most accurate to detectchanges in the methylation pattern of the MEREsequence as determined by the MethPrimer program(Li and Dahiya, 2002). For each CG, CNG, and CHHpresent in the sequence and for each time point of theexperiment, we determined the percentage of methyl-ation (percentage of C unconverted to T; Fig. 7A;Supplemental Fig. S4B). The results obtained for thetwo lines (Jemalong and R108) are similar (Fig. 7A;data not shown) and indicated that the MERE1 cyto-sines are highly methylated before the beginning of theexperiment and after 4 d of culture, when the expres-sion of the element is still very low (Fig. 7). A significantdecrease of the methylation status can be detected after7 d using the 2HA gDNA (14 d for R108), correspond-ing to the peak of induction of the element as shownby northern blot. At 14 d (21 d for R108), the percentageof mCG methylation increased again. This experimentshowed that the percentage of cytosine methylation ofthis region is inversely correlated with the activityof the retroelement, indicating a close connection be-tweenMERE1 expression and the methylation status ofthe CG and CNG cytosines present in its sequence. TheCHH residues that were highly methylated at the

Figure 7. Methylation status at theMERE1 locus. Thegraph represents the level of methylation (% methyl-ation; y axis) at the different C residues in thesequenced fragment. The DNA methylation levelwas determined using the bisulfite sequencingmethod and presented for each different sequencemotif: CG, CNG, and CHH (N = A or T; H = A, T, orC). The position in the sequence indicates C residues.For complete sequence analysis, see SupplementalFig. S4A. A, Percentage methylation of the CG andCNG residues present within the analyzed sequence.B, Percentage methylation of the CHH residues.These residues are divided into two groups: thosewith 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 CHH112and CHH145 from the second group (,20%) areshown.





beginning of the experiments also showed a transientdemethylation. However, this is not the case for theresidues that were much less methylated (Fig. 7B).

MERE1-1 Can Insert into Genes

In order to compare the nature of the sequencesflanking new MERE1-1 insertions and the ones of thewell-described Tnt1 tobacco element, 100 MERE1flanking sequence tags (FSTs) and 288 Tnt1 MedicagoFSTs were analyzed. The flanking sequences wereclassified in three classes: CDS (coding sequences),repetitive (repetitive elements), and unknown when atarget sequence could not clearly be identified (Fig.8A). Within the 100 MERE1-1 FSTs, we could identify33 ESTs using BLASTn analysis (http://compbio.dfci.harvard.edu/tgi/; E value of 10210). Similarly, aBLASTx analysis (E value of 10210) showed that 33sequences belong to the CDS class and nine to therepetitive elements class. The same analysis using 288Tnt1 FSTs shows nearly the same results: 28% of the288 FSTs belong to the CDS class and 6% to therepetitive elements. These results are in agreementwith those of Tadege et al. (2008), who showed using alarger population that 34.1% of the Tnt1 inserts are inORFs, despite CDS representing only 15.9% of the M.truncatula pseudogenome. This result indicates thatcoding sequences are tagged at a similar frequency byMERE1-1 and Tnt1. Moreover, the analysis of thesequences flanking the two retroelements indicatedno clear consensus target site for the two elements (Fig.8B). The GC composition of the 50 bp downstream ofthe two elements, including the TSD sequence, isroughly 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. Inaddition, at position TSD+3, G and C frequency wasincreased to 29% and 41%, respectively, instead of17.5% each (Fig. 8B; Supplemental Table S3). Thus, asfor Tnt1, there is a modest preferences for GC versusAT 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, whichshow root hair branching, nsp2-4, similar to the M.truncatula nfp mutant (Amor et al., 2003), is character-ized by an absence of root hair branching under ourtest conditions. Different genetic background (R108versus Jemalong) might explain the different root hairresponses. In line with these differences, we observedthat R108 wild-type plants showed reduced curlingand branching compared with the Jemalong wild-typeplants 2 d after infection, a time point when thebranching 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 Medicagoretroelement (MERE1) that belongs to a family com-posed of five to 10 members in the M. truncatulagenome. Our study using M. truncatula ecotypes ofdifferent geographical origin shows a constantMERE1copy number. This suggests a relative stability of thiselement copy number throughout the Medicago genus.

Figure 8. MERE1-1 has no targetsequence site specificity but tendsto insert within the ORF. A, Distri-bution of the genomic sequencesflanking newly transposedMERE1-1(100 inserts) and Tnt1 (288 in-serts). CDS are indicated in black,repetitive sequences in gray, andundetermined sequences in white.B, Target site sequence analysis. Atotal of 157 sequences were ana-lyzed for the MERE1-1 element and1,725 sequences for the Tnt1 ele-ment using the programWebLOGO3.0. The size of the letters indicatesthe frequency of each base pair. Thepositions of the TSD are indicatedby the arrows.

Rakocevic et al.




Three of the five copies, MERE1-1, -3, and -4, arecomplete LTR retroelements, with complete GAG-POLcoding regions. Surprisingly, only one of these fiveelements (MERE1-1) was active during tissue culture.The two other members with intact coding capacitywere stable under our experimental conditions.BLAST analysis and PCR experiments indicated thatMERE1-like elements are present in other legumespecies like L. japonicus and Cajanus cajan, but we didnot detect related elements in Arabidopsis. Interest-ingly, the U5 region of the LTRs is highly conservedbetween the elements in L. japonicus andM. truncatula,whereas the U3 region, which contains the promoterregion, is more variable. This more variable sequenceof the promoter region may represent an evolutionaryadaptation to control the activity of the retroelement(see below).Interestingly, the sequenced part of the genome

contains only five solo LTRs, which have significantsequence differences from the full-length MERE1LTRs. This suggests that other MERE1-1-like elementsmay exist in the nonsequenced part of the genome, butit 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 de-tected in a M. truncatula R108 background. Our workdemonstrated that transposition occurred in 40% ofthe R108 regenerated plants from a small mutantcollection, with an average new copy number of oneelement per plant in the total collection, indicating amild activity of the element in this background duringtissue culture. The stability of theMERE1 retroelementcopy number in M. truncatula (and L. japonicus) mightresult, as for Tnt1 (Grandbastien et al., 1994), from along coevolution of this particular family of retroele-ments with its host organism. Interestingly, this ele-ment was also found active in the M. truncatulaJemalong 2HA line used to generate the MedicagoTnt1 insertion mutant collection (www.eugrainlegume.org). In this line, theMERE1 element was also activatedby the in vitro tissue culture procedure, used primarilyto activate the Tnt1 element. This experiment suggeststhat the two retroelements might be activated under thesame tissue culture conditions and at the same time inthe mutant plants.The analysis of the MERE1 integration site se-

quences showed that there is no hot spot for integra-tion and no consensus sequence, except an increase ofG/C residue in the TSD+3 position, for MERE1 inte-gration site. In addition, the analysis of 100 insertionsite sequences showed that, like Tnt1 (Tadege et al.,2008),MERE1 inserts twice as much as expected insidegenes in M. truncatula.

The MERE1 Activity Is Detected during Tissue Culture

The induction of MERE1-1 during tissue culturereaches its maximum at different time points depend-

ing on the genotype used (7 d for 2HA versus 14 d forR108). A plausible explanation for these differencescomes from the genetic background and the regener-ation history of both genotypes. R108 and 2HA weresubjected to several cycles of somatic embryogenesison different growth media to select highly regenerableplants (Trinh et al., 1998; Rose et al., 1999). 2HA,therefore, will have a different regeneration capacityon a R108 medium, which was used herein to inves-tigate MERE1 induction during tissue culture. Thus,besides being genetically different, medium-dependentcallus growth may contribute to the observed shift inMERE1-1 induction.

Plants with new MERE1-1 copies did not show anytransposition activity in their T2 generation, suggest-ing that the MERE1-1 activation was restricted totissue culture. The absence of transposition in furthergenerations suggests that this might be the conse-quence of an increase in methylation, thus silencingand stabilizing the MERE1 retroelement in regener-ated plants. Transposition activity in the course oftissue culture could not be induced by stress-related orplant growth hormones, since MERE1-1 expressionwas not detected when either hormone was addedseparately to the regeneration medium (see “Results”).Our study indicated that only MERE1-1 transposes,since no copies of the additional MERE1 could bedetected. The most likely explanation is that the re-maining full-length MERE1 elements (MERE1-2, -4,and -5) have lost their coding capacities because of theincomplete ORF or aging restricts their transposition,as they have sequence differences between the 3# and5# LTRs. The identity between 5# and 3# LTR sequenceshas been previously reported to be correlated to theactivity of retroelements in mouse (Ribet et al., 2004).Those authors have shown that mobilization of MusDtransposon is correlated to 100% identity between itsLTRs, which is consistent with their recent transpositionand them being active. More recently, Maisonhauteet al. (2007) have shown that the newly inserted 1,731LTR retrotransposon copies in Drosophila show 100%identity compared with older copies. An exception isMERE1-3, which was not expressed (no cDNA de-tected) and was not recovered in any of the mutantscreens despite having an intact ORF and identicalLTRs. This suggests that MERE1-3 might be the targetof an epigenetic regulation. Its insertion in a particularregion of the M. truncatula genome might triggergenomic position effects that could result from a singlephenomenon or a combination of diverse epigeneticphenomena such as DNA hypermethylation, histonemodifications, or any other evolution of the chromatinstate. Epigenetic regulation of retrotransposons is aknown phenomenon reported in rice, Brassica rapa,Drosophila, and many other organisms (Cheng et al.,2006; Eickbush et al., 2008; Fujimoto et al., 2008). Wecannot know if the other copies detected in this anal-ysis as cDNA and gDNA fragments represent MERE1functional elements. Further investigations will shedlight on the underlying mechanisms responsible for





the differential regulation of MERE1 copies in M.truncatula.

We could not detect new copies of the Lotus MERE1-like element in the progeny of transgenic Lotus plantsregenerated in vitro (data not shown). Interestingly,transposition could also not be detected in Arabidop-sis transgenic plants transformed with the R108MERE1-1 element isolated from the MtNSP2 locus(data not shown). It should be noted that both trans-genic Arabidopsis plants and regenerated L. japonicusplants were produced using transformation/regener-ation methods not based on somatic embryogenesis.This suggests that transposition of MERE1 is depen-dent on the genetic background used, on tissue cultureconditions, on the combination of both, or on otherunknown regulatory mechanisms. Such genotype-and tissue culture-dependent transposition activitywas described for Tnt1 in the M. truncatula 2HA line(Iantcheva et al., 2009). It might thus be interestingto regenerate MERE1-containing Arabidopsis or L.japonicus plants using an embryogenesis-mediatedmethod in order to know if this procedure is crucialfor the activation of these elements.

Interestingly, a phylogenetic analysis includingmost of the plant copia retroelement clades indicatesthat several tissue culture-activated plant copia ele-ments (MERE1-1, Tnt1, and Tto1) belong to the samesubfamily of the copia clade 1 (Supplemental Fig. S2),suggesting that they may have retained common reg-ulatory mechanisms that allow their activation duringtissue culture.

Transcription of MERE1 Correlates withPartial Demethylation

Methylation is generally correlated with the repres-sion of retroelements (Martienssen and Colot, 2001).We observed a clear correlation between the expres-sion of the element and a transient diminution of themethylation status of the MERE1 element during theearly steps of our in vitro experiments. The transientreduction of the methylation was particularly clear forthe CG residues and for one of the two methylatedCNG residues present in the analyzed sequence. Thehighly methylated CHH residues also showed a tran-sient demethylation. Thus, there is a clear correlationbetween the methylation status of the element and itstransposition activity during in vitro culture. Suchtissue culture-induced demethylation was previouslyreported in soybean (Glycine max) and maize (Zeamays; Quemada et al., 1987; Kaeppler and Phillips,1993). Although decrease in methylation is often as-sociated with tissue culture, it is still unclear why thishappens. We can only hypothesize an adaptive re-sponse of the plant to tissue culture stress conditionsthat allows a transcriptional activation of appropriategenes. Reduced methylation of MERE1 suggests thatour in vitro culture conditions (somatic embryogenesis-mediated plant regeneration) might induce importantchanges in the methylation status of the retroelement,

probably reflecting a transient demethylation of largeregions of the Medicago genome that might be respon-sible for the activation of the MERE1 retroelement. Onemight speculate that this could also be the case for thetransposition of Tnt1. This is a likely scenario, since itwas shown in Arabidopsis that Tnt1 silencing is re-leased in decrease in DNAmethylation and polymerase IVamutants (Perez-Hormaeche et al., 2008). Furthermore,those authors report a negative correlation betweenTnt1 copy number and its silencing, which was shownto be associated with short interfering RNAs, suggest-ing 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 (2HA3-9-10-

3), 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 character-

ized 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 en-

vironmentally 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 R108-

derived T-DNA mutant collection (Brocard et al., 2006) inoculated with

Sm1021. The nodulation test was carried out in the greenhouse with approx-

imately 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)

Rakocevic et al.




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.

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 (Ase-

adap1 [5#-CCCCTCGTAGACTGCGTACC-3#] plus Ase-adap2 [5#-TAGG-

TACGCAGTCTACGA-3#]) were ligated (T4 DNA ligase; Fermentas) at 16�Covernight. 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#-AGCCCTTTTGTCAAACATGTA-

TTA-3#), MERE2 (5#-AATGTTGGCACCGAAAATCTGAATGGT-3#), MERE61

(5#-AACAAAAGGTGACTTTATGGCTT-3#), MERE62 (5#-CAACAAAATG-

TGGCTTTACTTGT-3#), MERE63 (5#-CAACAAAAGTGGCTTTACCACT-3#),MERE64 (5#-CAACATGATTTGACTTGGCACA-3#), MERE51 (5#-GCCTCA-

ACCATTTTCATTTATTACCAT-3#), and MERE52 (5#-TGCCTCAACCAMTY-

SMATTTAWGGCA-3#).

Northern Blot

Total RNAwas extracted from 2 g of plant material according to Kay et al.

(1987). The RNA concentration was measured with a spectrophotometer (ND-

100; 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 geneMtc27was used as a

control (TC 85211; Verdier et al., 2008).

RT-PCR

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 gDNAwas 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#-AGTCTCTCTCTGCGGCT-

GAG-3#] and EF1b [5#-CGATTTCATCGTACCTAGCCTT-3#]) was used in

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 forMtEF1a and 30 cycles for other genes.

Amplification products were analyzed using 1.5% agarose gel electrophoresis.

Segregation Analysis

For the segregation analysis, we used primers NSP2-5# (5#-CCGGCAG-

GCGATTAACCTCCTT-3#), NSP2-3# (5#-GCATCCTATAAATCAGAATCT-

GAA-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 con-

verted (+)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#-TAGCAACTGGAGTT-

GGCAAGGTAGAGAG-3#) and BiWT2 (5#-GAATGAAAAGCTGGATTCC-

TTGCAA-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).Thealign-

ments of the GAG-POL regions were conducted using ClustalX (Thompson

et al., 1997). Relationship tree building was conducted using MEGAversion 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

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.





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 Valerie 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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mere1, a low-copy-number copia-type retroelement...mere1, a low-copy-number copia-type retroelement...

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