supplementary information · expressing evd: epi12 (f9) and epi35 (f9) previously described in...

Reconstructing de novo silencing of an active plant retrotransposon.

Arturo Marí-Ordóñez, Antonin Marchais, Mathilde Etcheverry, Antoine Martin, Vincent Colot and Olivier Voinnet.

SUPPLEMENTARY INFORMATION:

Supplementary Figure 1: EVD vs ATR expression and LTR methylation.

Supplementary Table 1: Figure 1 and Supplementary Figure 1 bisulfite sequencing raw data.

Supplementary Figure 2: Arabidopsis gynoecium and ovule development.

Supplementary Figure 3: EVD in situ hybridization in WT and two additional epiRILs.

Supplementary Figure 4: Suppression of silencing assay in N. benthamiana.

Supplementary Figure 5: GAG RNA binding domain conservation and EVD polyprotein product

production in EVD constructs.

Supplementary Figure 6: Mapping of 21-22-nt siRNAs to EVD in epi15 F14 and EVD copy

number in epi454 and 438 F8.

Supplementary Figure 7: Bisulfite sequencing dot-plots of EVD LTR in epi15 generations and

EVD copy number and DNA methylation in epi439 generations.


Supplementary Figure 8: Correlation of LTR 24-nt siRNAs abundance with EVD copy number

and 21-24-nt siRNAs mapping correlation on EVD.

Supplementary Table 3: Figure 5 bisulfite sequencing raw data.

Supplementary Figure 9: Bisulfite sequencing dot-plots of RPP4 Solo-LTR in epi15 generations.


Supplementary Figure 10: EVD life cycle in Arabidopsis.

Supplementary Table 5: List of primers.

Nature Genetics: doi:10.1038/ng.2703

(bp)

-578

-448 -415

-163

ATR (AT1G34967)

EVD (AT5G17125)

1026 bp

448 bp

448 bp 578 bp

415 bp 163 bp BglII BglII

BglII

RT+

WT met1 epi15 F8F11O6(Chr1)

F2K13(Chr5) gDNA

WT

epi1

5 F8

m

et1

EVD (AT5G17125) 5’LTR ATR (AT1G34967) 5’LTR

0% 10% 20% 30% 40% 50% 60% 70% 80% 90%

100%

WT met1 epi15 F8 WT met1 epi15 F8

EVD ATR

% m

ethy

late

d cy

tosi

nes

CG CHG CHH

a b

c

Supplementary Figure 1

Supplementary Figure 1: EVD vs ATR expression and LTR methylation.

(a) Restriction fragment length polymorphism (RFLP) analysis of RT-PCR products

shown in Figure 1a. genomic DNA (gDNA), and PCR products obtained from F11O6

BAC (comprising chromosome 1 ATR, AT1G34967, copy) and F2K13 BAC (compris-

ing chromosome 5 EVD, AT5G17125, copy), were digested with BglII to determine

whether transcripts originated from EVD or ATR. (b) Bisulfite sequencing-based meth-

ylation analysis at the EVD and ATR 5’LTR in WT, met1 and epi15 F8. The 5’LTR of

AT5G17125 (EVD) and AT1G34967 (ATR) was specifically amplified using a primer

inside the LTR and a specific primer outside of the LTR 5’ end. Cytosine methylation

levels are given at CG, CHG and CHH sites. Error bars represent the 95% confidence

limits given by the Wilson score interval (see Supplementary Table 1 for details). (c)

Dot-plots depiction of bisulfite sequencing data obtained for individual clones used to

generate the results shown in Figure 1b, Supplementary Figure 1b and Supplementary

Table 1. Filled circles indicate methylated cytosines and empty circles unmethylated

cytosines.

CHH CHG CG

WT

epi1

5 F8

m

et1


L2

L1

MMC

MMC Sync FM

b

ad

medial domain: fused lateral margins

niamod l ar et al

cmm ab

p p

a

Supplementary Figure 2: Arabidopsis gynoecium and ovule development.

(a) Scheme of a cross-section of the Arabidopsis gynoecium. The gynoecium is formed

by the congenital fusion of two carpels (modified foliar organs that bear the ovules)

through their lateral margins (cmm, yellow). Placental tissues (p, pale yellow), from

which ovules arise (see b), differentiate from the cmm at the intersections of lateral and

medial domains21-23. ab, Abaxial lateral domain (green); ad, adaxial lateral domain

(blue); abaxial medial domain (orange). Adapted from ref. 23. (b) Ovule development

in Arabidopsis. Ovule primordia emerge from the placentas, where a somatic cell from

the sub-epidermal L2 cell layer (in red) differentiates into the megaspore mother cell

(MMC) that undergoes meiosis, forming a syncytium (sync) of haploid nuclei, of which

three degenerate, leaving a single haploid product: the female megaspore (FM). The

FM then gives rise to the female gametophyte. Adapted from ref. 24.



epi15 WTa

3

1 2

5

7

9

4

6

8

10

WT epi15 epi12 epi35 b

21

15141312

111098

75

43

6

WT non transf. WT pLTR:GUS-GFPc


Supplementary Figure 3: EVD in situ hybridization in WT and two additional

epiRILs.

(a) In-situ hybridization study of EVD transcript accumulation, using an antisense

probe, in developing embryos of WT and epi15 F10. Longitudinal sections of embryos

at globular- (1-2), heart- (3-4), mid-torpedo- (5-6), late-torpedo- (7-8) and mature

green-stage (9-10). (b) In-situ hybridization study of EVD transcript accumulation,

using an antisense probe, in various tissues of WT, epi15 F10 and two other epiRILs

expressing EVD: epi12 (F9) and epi35 (F9) previously described in ref.18. Longitudinal

section of the SAM region from a young Arabidopsis rosette (1-4). Longitudinal sec-

tions of flower bud (5-7). Gynoecium transversal sections at floral developmental stage

8 (8-11). Longitudinal sections of ovule primordia at floral developmental stage 12

(12-15). (c) Longitudinal section of plant rosettes at the SAM region of non-transgenic

WT and pLTR:GUS-GFP transgenic WT plants, following GUS-staining. Bars = 50 ȝP�

for (a) 1-4, (b) 12-15; 100 ȝP�IRU�(a) 5-10, (b) 1-7 and (c); 10 ȝP�IRU�(b) 8-11.


DCL4DCL2

dsRNA

21nt-siRNA

(A)n

AGO1

RDR6

AGO1

GFP GFP + P19

DCL4DCL2

dsRNA

21nt-siRNA

AGO1

RDR6

P19

DCL4DCL2

dsRNA

21nt-siRNA

AGO1

RDR6

GFP + P19m

(A)n (A)n

AGO1

P19m

@EVD

GFP + P19 GFP + P19m GFP + EVD 4 dpi 8 dpi 4 dpi 8 dpi 4 dpi 8 dpi

@GFP

@U6

#�·*$*�

@GFP

EtBr

mR

NA

siR

NA

s

a b

-4.2kb

-21nt -24nt

-21nt -24nt

-0.7kb


Supplementary Figure 4: Suppression of silencing assay in N. benthamiana.

(a) Suppression of silencing assay by Agrobacterium-mediated transient expression in

N. benthamiana leaves. Agrobacterium tumefaciens, infiltrated into plant leaves medi-

ates transfer of transgenes into plant cells, allowing ectopic expression of the genes of

interests, but it rapidly decreases 3-4 days post infiltration (dpi) due the onset of

PTGS30. This feature of the system has been routinely used to characterize proteins

with VSR activity by co-infiltrating the protein of interest with a GFP reporter transgene

acting both as a trigger and a target of PTGS30 . In the scheme, when infiltrated alone,

GFP protein levels (visualized under UV illumination) are reduced due to the action of

PTGS; however co-infiltration with the VSR P19, prevents the onset of PTGS in the

infiltrated area allowing significantly higher levels of GFP accumulation. P19 acts as a

head-to-tail homodimer that specifically binds 21-nt small RNA duplexes, preventing

their loading into AGO. This property is abolished in the point mutant P19m, which

dimerizes but is unable to bind small RNAs. (b) Analysis of p35S:EVD transient

expression in N. benthamiana. The potential suppression of silencing activity of EVD

was tested by co-infiltration of EVD constructs (Figure 2g) with the p35S:GFP reporter

construct. p35S:GFP co-infiltrated with p35S:P19 or its mutant version p35S:P19m

were used as controls for positive and negative suppression activity, respectively.

Northern analysis of High-molecular weight (mRNA) and low-molecular weight RNA

(siRNA) was carried out on samples extracted from infiltrated leaves at 4 and 8 dpi.

Ethidium bromide (EtBr) staining of ribosomal RNA and detection of U6 are shown as

loading controls.


MOCK

HA-EVD-FLAG

HA-EVD¨R

BD -FLAG

MOCK

HA-EVD-FLAG

HA-EVD¨R

BD -FLAG

@HA @FLAG

-35 kDa

-55 kDa

Coom. Coom.

c35S GAG PR IN RT-RNase

35S GAG PR IN RT-RNase

...YKNEDRKLLTCDHCKKKGHTKDKCWLLHPHLKPA...

...YKNEDRKLLT-----------------------------WLLHPHLKPA...

HA-EVD-FLAG

HA-EVD6RBD-FLAG

HA

HA

FLAG

FLAG

b

130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 305 315 325 374 384 394 404

MPMV/1-275SRV-1/1-275SERV/1-274JSRV/1-280SMRV-H/1-285MMTV/1-280LPDV/1-269RSV/1-298HERV-K10/1-287HIV-2/1-243SIVMAC/1-245HIV-1/1-244SIVAGM/1-248SIVMND/1-253EIAV/1-242FIV/1-227VMV/1-227SA-OMVV/1-226CAEV/1-228BIV/1-240STcLV2PP1664/1-213HTLV-2/1-213HTLV-1/1-213KoRV/1-251GALV/1-252MdEV/1-252PERV-MSL/1-255BAEVM/1-241RCHO-K1/1-237MuLV/1-250FeLV/1-241REV/1-227HERV-E/1-235RTVL-Ia/1-228WdSV/1-209Cereba/1-255CRM/1-261Galadriel/1-235Monkey/1-159Legolas/1-269Tma/1-118Retrosat-2/1-273pCretro3/1-221pCretro6/1-229Pyggy/1-234Real/1-233Cgret/1-271Pyret/1-248Skippy/1-245Maggy/1-241Dane-1/1-218Cer5/1-245Cer6/1-262Cer4/1-250Peabody/1-273Beetle1/1-240Cft-1/1-259Sushi-ichi/1-253marY1/1-249Reina/1-236Gloin/1-227Ifg7/1-159Micropia/1-240Blastopia/1-226MGLR3/1-246Grasshopper/1-279Cigr-1/1-257Mag/1-214SURL/1-245Osvaldo/1-267Cer1/1-222RIRE2/1-236Ogre/1-290Tat4-1/1-249Tft2/1-239Ty3-1/1-255Circe/1-277Skipper/1-230Athila4-1/1-229Cyclops-2/1-234SIRE1-4/1-314Mdg3/1-227Endovir1-1/1-299Oryco1-1/1-279Poco/1-257Batata/1-237Tto1/1-236V12/1-239RTvr2/1-233Tnt-1/1-238Tork4/1-231Xanthias/1-2351731/1-230Tricopia/1-242Yokozuna/1-207Copia/1-238Hopscotch/1-276Retro!t/1-281Koala/1-286Hydra1-1/1-227Hydra1-2/1-237CoDi6.3/1-266CoDi6.2/1-287Zeco1/1-231Olco1/1-230Cico1/1-224GalEa1/1-271CoDi6.5/1-296Mtanga/1-211CoDi6.4/1-289Oryco1-2/1-270Humnum/1-235PyRE1G1/1-243Koco/1-241Sto-4/1-262Fourf/1-234Osser/1-257Melmoth/1-256TSI-9/1-297Tse3/1-214Ty4/1-212EVADE/1-265

QQAGNWDF DML T GSGNYS S T DAQMQYDPGL F AQ I QAAAT KAWRKL P VKGDPGAS L T GVKQGPDEP F ADF VHRL I T T AGR I F GSAEAGVDYVKQL AY ENANPACQAA I R - - P Y RKK - - - T DL T GY I RL CSD I GP S YQQGL AMAAAF SGQT VKDF LQQAGNWDF DML T GSGNYS S T DAQMQYDPGL F AQ I QAAAT KAWRKL P VKGDPGAS L T GVKQGPDEP F ADF VHRL I T T AGR I F GSAEAGVDYVKQL AY ENANPACQAA I R - - P Y RKK - - - T DL T GY I RL CSD I GP S YQQGL AMAAAF SGQT VKDF LQQAGNWDF DML T GSGNYANT RAQMQYDPGL F SQ I QAAAT KAWRKL P VKGDPGAS L T AVKQGPNEP F SDF VHRL MT T AGR I F GNAE T GVDY VKQL AY ENANPACQAA I R - - P Y RKK - - - T DL T GY I RL CSD I GP S YQQGL AMT AAF SGQT VRDF LRQQG I T S Y EML I GEGPYQAT DT QL NF L PGAYAQ I SNAARQAWKKL P S S S T KT EDL SKVRQGPDEP YQDF VARL L DT I GK I MSDEKAGMV L AKQL AF ENANSACQAAL R - - P Y RKK - - - GDL SDF I R I CAD I GP S YMQG I AMAAAL QGKS I KE V LRP P PKDGP L K I PGAS P YQNNDKQAQF P PGL L T Q I QSAGL KAWKRL PQKGAAT T S L AK I RQGPDES Y SDF V SRL QE T ADRL F GSGES E S S F VKHL AY ENANPACQSA I R - - P F RQKE - L S T MSP L L WYCS - - - - AHAVGL A I GAAL QNL APAQL LKRKK - V S L DML L GT GQF L S P S SQ I KL SKDV L KDV T T NAV L AWRA I P P PGVKKT V L AGL KQGNEE S Y E T F I S RL E EAV YRMMPRGEGSD I L I KQL AWENANS L CQDL I R - - P I RKT - - - GT I QDY I RACL DAS PAV VQGMAYAAAMRGQKYS T F V- - V T V AV L KG - SDPAMAT PQL QAARMR - GRE I QASCQASV S I CGGRT VGKRT DPWT KV T QGL GEP F L S F AERL L NAY EKSQL P EAAKNAV F RDCVKQQGNML T QQYML GAPS T ENT - - AE L V KY L L KRENEG - - AAQGNAAA I V AAL K - - - - - -E RT NL DRL KGL ADGMVGNPQGQAAL L RPGE L VA I T ASAL QAF RE VARL AE PAGPWAD I T QGPS E S F VDF ANRL I KAV EGSDL P P T ARAP V I I DCF RQKSQPD I QQL I RAAP S T L T T - PGE I I KY V L DRQKT AP L T DQG I AAAMSSA I QP L VMAV- P P V I DADQL L G I GQNWST I SQQAL MQNEA I EQVRA I CL RAWEK I QDPGS T CP S F NT VRQGSKE P Y PDF VARL QDVAQKS I ADEKAGKV I V E L MAY ENANPECQSA I KP L KGKV PAGSDV I S E Y VKACDG I GGAMHKAML MAQA I T GV V L GGQVRE PRGSD I AGT T S T V E EQ I QMF RPQNPV P VGN I Y RRWI Q I GL QKCVRMYN - P T N I L D I KQGPKE P F QS Y VDRF YKS L RAEQT DPAVKNWMT QT L L VQNANPDCKL V L KGL G - 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- - - P - E DT L E EKMYACRD I GT T KQKMML L AKAL QT GL AGP F KGPRP L P Y F T AAE I MG I GL T QEQQAARF APARMQCRAWY L EAL GKL AA I KAKS PRAVQL RQGAKEDY S S F I DRL F AQ I DQEQNT AE VKL Y L KQS L S I ANANAECKKAMSHL K - - - - P - E S T L E EKL RACQE I GS PGYKMQL L AEAL T KVQVVQSK- - - - - - V L T VDQ I MGVGQT NQQASANMDQARQ I CL QWV I T AL RS VRHMSHRPGNPML VKQKNT E S Y EDF I ARL L E A I DAE P V T DP I KT Y L KV T L S Y T NAS T DCQKQMDRT L GT RVQ - QAT V E EKMQACRDVGSEGF KMQL L AQAL RPQGKAGHK- - - - - - V L T VDQ I MGVGQT NQQASANMDQARQL CL QWV I T AL RS VRHMSHRPGNPML I KQKNSE S Y EDF I ARL L E A I DT E P V T DP I KT Y L KV T L S F T NAS T DCQKQMDRV L GT RVQ - QAS V E EKMQACRDVGSEGF KMQL L AQAL RP PRKEGKQ- - - - - GGL T VDQ I MGVGQT NQAAAANMDQARQ I CL QWV I NAL RAVRHMAHRPGNPML VKQKT NE P Y EDF AARL L EA I DAE P V T QP I KDY L KL T L S Y T NASADCQKQMDRT L GQRVQ - QAS V E EKMQACRDVGSEGF KMQL L AQAL RPGKGKGNG- - - - - - - - - T DE I I GKGL S SAEQAT I S V ECRE T F RQWV L QAAMEVAQAKHAT PGP I N I HQGPKE P Y T DF I NRL VAAL EGMAAPE T T KE Y L L QHL S I DHANEDCQS I L RP L G - 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KS KT L GDL VREAEK I F NKRE T P E ERE ER I RRE T E EKE- - T QL PNV I DE T F P L T RPNWDF AT PAGREHL RL YRQL L L AGL RGAARRP T NL AQVKQVVQGKEE T PAAF L ERL KEAYRMY T P YDP EDPGQAAS V I L S F I YQS S PD I RNKL QRL EGL - QGF T L SDL L KEAEK I Y NKRE T P E ERE ERL WQRQEERD- - V T DERE I E AQF PAT RPDWDPNT GRGNDNL ERYRQ I L L RGL RAAARKP T NL SK I T E V RQGADES P T AY L ERL YQAYRT WSP I DPRAP ENQAA I V I QF V SQSAPD I RKK I QK I DGF - QGKS L S E L VA I AQKV F DQREDPAKAT HE L T QKMAKV L- - QNPQEY VRT QL PGT DPQWDPNR - EDMQRL NRYRKAL L EGL KRRAQKAT N I NKV S E V I QGKE E S PAKF HERL CEAYCMY T P F DPDS P ENQRM I NMAL V SQS T ED I RRKL QKKAGF - AGMNT SQL L E I ANQV F VNRDAASRKE T T RMN - - - - - -- - - - - - T QKL SKAF D I QQEKDEGP I RF L DRL KEQMKQY T GL NL EDP L GQRML K I HF V T KGWPDVSKKL QKAQKVY VRRDREKQKQKSKPML S T F QQVAPNP YAT KGF QGARN - - - - - - - - - - - Y KRSQASQT QF RE T KP SARGPKS T F PRP PKE- - - - - AF T Y KP PQT AHE Y VKHAE I I F KNNSGL EWQHAT V P F I NMVVQGL P PKV T RS L MSGNPDWS T KT I PQ I I P L MQHY L L QSRQDAK I KQT P L V L QL AMPAQT MNGNKGYVG - 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- - I E E E RY L NREK - AP I RNNQS T KPADKKRKF DKVDNT KSDAKT GECV T CGRQAD I SWADF VAE F NAKY F PQEAL DRMEAHF L E L T QGS - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -P GT E V T WAE F CRS F RKAQVPDGVVAQKKRE F RAL HQGNRT V T E Y L HE F NRL ARYAP EDVRT DAEKQEKF MAGL DDE L T NQL I SGDYADF ERL VDKA I RQEDQRNKMDRKRKAAQF R - APQGSHQRPRF T PGQQGGP T T M I V RQHRP F NS SNF HQ- - - - - I RL RCS PAVA I H I T SCE T AKSAWDT L KNMYAT QGT MAK - - - V T L V RRL QKY EMSEGADMEDE L RKL T EMRAEAAEAGG - - K I T DEDF AF N I L S AL P P SWES F VAAQN - - - - - - - - - - - NV E K - - - S SDV I GRV L S ENL RRQSK - - GDT T- - - - - I RL RV SDAV L V Y I S AAT T AKEAWDT L MQT F QPSGMYSY - - - I L AY RKF YQT RCPDGGD I P EHL GQMRRL RDE I HT I AGT AR I SDRE F AF AL L I S L PDSWDQF I QA I D - - - - - - - - - - - AT ADNL T SAQV I AR I L QEDKRRKEREGAKGD- - - - - S I DE L WHF MDQQYDDPHQKSKAL DRL L NL RQGKL S VRDYHME F NRL E I QSGERF GEAAKKSMF L KGL HT KL QEAL AT VDEDL - S Y EQL I NKA I RT SDNL Y - - - - - - - - - - - - - - - - - - RV S L T AR - ARQGHT L AEQS T PKT T QREAS - -- - - - - T V VGL WEHMDT HF QDVHQQQRAL NKL RHL KQGRRP I RDY V S E F NQL RV E SRQQF S P V VAREMF S EGL RE E L QKL ML HT PKNG - S L KE YMDKA I E L SDDL Y - - - - - - - - - - - - - - - - - - R I QL HGRNQRS T AHDGAQNHPRAVQREAS - -- - - - - - F DGY VKAL QS L F L DPDEKRQAERDL SNL RQNK - S AT L Y AAE F RRL AAR - - L DMT DE SKV F AF YQGL KDDVKDEMAKVDT P P - S F L DY V E YA I K I DNRL F E RRKEKGEKR - - - - - - - - QNPNSGRKYQWQPQQRF NNQRNDNRPRNNWN- - - - - - F AGYKRT F RS L F QE PDKKRQAERNL ANL RQT K - S ASAY T T E F KRL AT R - - L D I T E KT K I L QF YQGL KL R I KDE V SKL DRP E - DF L E Y V E L A I KL DNR I Y E RRQEKQGGQR - I F V T L I RQT NT GRKYQHPQP T YHRNNGGWQQPRHMAP- - - - - GRHEMEEAL KMAF GT I DEKGQAERK I KT L KQT G - S AS T L GV E F L QL ASK - - L PWDQDV L MS F F F DAL KEQVQQE L WEKDRPR - T L V E Y I NMAVK I DDRQF AWRT - - - - - - - - - - - - - - RNSRGNKGRQDNKPRYHANQGRT RQT D - - - -- - - - - DY T DL L QY L E EAF GDPDHVQNAQNKL YAL KQRNVDF AE Y L S E F QRL S L EG - - EMP EDAL P P L L F QG I S E E L QDML L HNPAP SRQYHE F T RHL QS L DNRYRQHQQYKN - - - - - - - - - - - RQT R - - - - - T P RAAAP PARAAPRT QP - D I P R- - - - - DY ED I L D I L DQAF GDPNCVNNARNE L F RF QQNNKE F GL F F AE F QHL AL EG - - EMP E E T L S T L L EQL I NRE L KGML MYNQPP T - DYHE F AKF L QE L ENRRRHYE I NL Q - - - - - - - - - - - S AS RNY PA I T RT AT SQL L RT NY T T L P RT I E N- - - - - S D I Y S I S F DDT V T AL T K I F GS T KS L MMRRQQCL Q I CRANGL SQDY L DY T NS I SDAV L DSKL S SMT SDEWS I F L F L RGL NS PGDEKAKL Y L MQYV EAS EKKNEKL KL S - - - - - - - - - - - DVHDEWMKF I QMHQQSK I V S VKP SKS SQQVD- - - - - T DF L G I P F T E T L QNL T KAF GSHRSMMMRRQQCL Q I S RANGES L DP L E Y SNRVGDAV L EAKL ASMS T DEWSV F I F L RGL DAPGDNPAKAY L MQF CE T AEKKGEKL KL S - - - - - - - - - - - D I HDEWI RF I QMKQQT KV V SAL P VKP T PQQA- - - - - CD I Y E V P F V T T V EAL EKAL GSHRS L M I RRQT CL HL SRT NCT S L DP L KWT NQ I S E AV L DAE L AKMS T SDWG I F L F L KGL DAPGDAQAKAY L MQF Y E T SDRKGEKL T L S - - - - - - - - - - - NMHDEWVRF L QT KSQT K I V SHS - - - - S P KT S- - E V I T WDV F RRE F MT KY Y P EDVRGKKE I E F L E L KQGNMSV T DYAAKF V E L SKF Y PHY T GAGAE F SKC I KF ENGL RS E I KKAVGYQK I R I F T E L VDSCR I F E EDNNAHYK I V SDRRGKQHQNRGKPYDAP VGKGKQGAAPAQRT SRGGAPAGYS- - K I KSWKKL KKKMEAKF L P S T Y T L DL YNQL SDL F QGT KNVE T Y I HE Y EKL MMKL DVQERE EQT MARF VKGL DRD I QKKL E L QS Y S T L DE L F RL AL KV EKHQKDKKKKV F SKG - - - - - - - - - - T S Y AKGGSS S Y VKP T S T S P F T P KS EAGSKF L- - ADGV F KS YNHL KHAMKSV F GVSNE I AT AVRV I QHL T QKT S T AE YAAKF QEYAQL T DWDDEAL QVMYRRGL KEHVKDE L MRDGRK I DGL GDL VQV T I DL DDKL Y ERAMERRYDSK - - - - - - - V SGKAGY T PGYDNRNRGF NDNYNKPKDKP Y Y- - - - - L S SDY SAF RRE F KAV F EHP T YGEDAASRL L AL QQGSRS VAE Y T L E F R I L AAE SRWGE T AL RSAYRRGL S EA I KDL I V RDRP S S L NE L I T L S L QMDERL RERRQERAQRAGGS T RQL SHRT S SAPDF S L T S T AAP P PH I L L QS PAHP S PR- - 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- - - - - - - - - - - - DDNL GPDQKNRKAS P V VCHF CSKPGRR I A- - - - - - E KQKY VQARSKM I GSAE L F L E S ECV SGY T E L KE L L I E E F SGS YNSAV I HKKL QDRKKKRE E T L HDY L L QMKK I AAL GE V E T VAL I T H I V NGL D I KKE YKGAML RCK - - - - - - - - - - - - T L KE L KQE F E I Y E S L N I V DKPN I QPKPKQ I- - - - DQ I T T YDG I MGHL RE E YKDT KL EDT ARDE L DKL I MS T KDKF M I F KNKF VKAGQSGL PKRSWKRE L HRRL P T NL RVAMV I YHQDT NV S F DAY VRT ADG I S YDL T KAYAS - - - - - - - - - - - RT EDKNKT KT T S I KKT T GGSV I P RAGGPARTRDGAYRY T DF L DY L ERT YDDPHKRAQAL AE L E T L KMKPGQS F AQF I A I F E RT L AT AGGL AWADEVRT NF L RF RV S PR I RE ACVGRGMGDGT Y L GAVA I Y RQVAQDL EA I E L DRRF GPHRAGAAT APRP PKDEDT PMT GVAAMGSRPNGGARGRR- - - - - - Y E E I I RT L RS V Y I KKKNN I Y S RY L L L S RQQSP E E S I P E Y I QE L KS L SRQCE F T AV EANQYRE E L T RDAF I DGL S S S V I RQRL L E VDS L DL MKAF E L ADS L DRAQKYS T SMGRNAAP T P S YAASAPQHQHEPKRSAPQHQYE E T VCRQQ- - - QL T Y ANAVAML AAHL QPKP S I L AERYKF RQRRQL NE S I ADY L T E L KKL SKNCE F GSS L DENL RDQMVCGL KS E I I RQRL F AE EKL E YKRAV T L AL S L EAAERDA I AV ER - - - - - - - - - - - - - T P I E E VNK I NF NECSRCGDRRHQAKDC I Y- - - - - - GE SMKL EKV L DKY E E YCMPKRNL T Y ERHRF F T RSQL EHE S VDQYAT E L RSRAQSCE F S S L KDS L I RDR I I CG I L DAGL RERL L RQDDL T L DKT L QVCRAAEQT RT QAQAL S SAGENAS I P VDVDS F SKT KKKF SGSKSKSKQSKPQDH- RHWRT WGT F VRS F QE F F F AEDY L EDL KDE VKRRKQMVDEP F K I YMV EMQT L MRP L RYGPDHEMKL I Y NNS I P DL RAYARP YQF QS L ME L MKL ADE F E E L ERDRERL RRL QRPART RL MAMEEDDGHEE EML RRGAL E E S PRPAPRT GAT GQRT- - - - - K I MDT DE I NHYDVCREM I I RHEWNVSK I NEKQCL NSKL S EKRGKVQNAENF VNQNT QNNF KP F S PNKAADNSRNSWNNNSQNNSAASQN I S REQSWKT I S V PQKHQN - - - - - - - - - - - P SDRCSDCQQRGWHMF WCSKKSKDNASQKCD- - - - - SWAE L RDHF I ANF QGT F ERPGT QYDL YNV I QKSGES L RDY I RRF S EQRNK I SD I T DDV I I AAF T KG I RHE E L VGKF GRKP PKT VKL MF EKANEYAKAEDAV T ASKQS - - - - - - - - - - - GP SWKPNKGT PAKGGGGSNNHKDRKRKP E E LMDMAPSRT QL QS L CQKSNES F KE YAQRWRE L AARVQPPML ERE L T DM I GT MGSCP F GS F SDV V I CGERT E S L I KT GK I QDVGSS S SKKP F AGAPRRREGE T NAVQHRRDQNR I E Y RQAAAV T I P APQPRQQQQQRVQQPQQQQQQRPYQPRQRM- - 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- - - - - - - - L CPRCKRGKHWANEC - - - - - - - - - - - -- - - - - - - - L CPRCKRGKHWANEC - - - - - - - - - - - -- - - - - - - - L CPRCKRGRHWANEC - - - - - - - - - - - -- - - - - - - - L CPRCKKGKHWARDC - - - - - - - - - - - -- - - - - - T T KCPRCKKGF HWASEC - - - - - - - - - - - -- - - - - - - - L CPRCKKGYHWKSEC - - - - - - - - - - - -- - - - - - GARCWSCGGAGHL ARDC - - - - - - - - - - - -- - - - - - RE RCQL CDGMGHNAKQC - - - - - - - - - - - -T T GRE P PDL CPRCKKGKHWASQC - - - - - - - - - - - -- - - - - - - QGCWKCGKPGH I MT NC - - - - - - - - - - - -- - - - - - - QGCWKCGKMDHVMAKC - - - - - - - - - - - -- - - - - - - KGCWKCGKEGHQMKDC - - - - - - - - - - - -- - - - - - - NKCL KCGKPGHL AKDC - - - - - - - - - - - -- - - - - - - KGCWNCGAMDHQKAQC - - - - - - - - - - - -- - - - - - - KVCF KCKQPGHF SKQC - - - - - - - - - - - -- - - - - - - KKCNKCGKPGHL AAKC - - - - - - - - - - - -- - - - - - - I I CHHCGKRGHMQKDC - - - - - - - - - - - -- - - - - - - I I CHHCGKRGHMQKDC - - - - - - - - - - - -- - - - - - - I I CHNCGKRGHMQKEC - - - - - - - - - - - -- - - - - - - QKCYHCGKPGHQARNC - - - - - - - - - - - -- - - - - - - - P CP L CKDPSHWKRDC - - - - - - - - - - - -- - - - - - - - P CP L CQEP SHWKRDC - - - - - - - - - - - -- - - - - - - - P CP L CQDP T HWKRDC - - - - - - - - - - - -GRP P L DKDQCAYCKERGHWAREC - - - - - - - - - - - -GRP P L DKDQCAYCKEKGHWAREC - - - - - - - - - - - -RKT P L E KDQCAF CKEKGHWAKDC - - - - - - - - - - - -NRT P L DKDQCAYCKERGHWARNC - - - - - - - - - - - -RRPKVDKDQCAYCKERGHWI KDC - - - - - - - - - - - -RGPHL DRDQCAYCKEKGHWAKEC - - - - - - - - - - - -RRPQL DKDQCAYCKEKGHWAKDC - - - - - - - - - - - -RK I P L GKDQCAYCKEKGHWVRDC - - - - - - - - - - - -GRP P L GKNQCAYCKE EGHWKKNC - - - - - - - - - - - -GCQS L QCNQCAYRKE I GYWKNKC - - - - - - - - - - - -- - - - - - - - KCYKCGRT AP F KREC - - - - - - - - - - - -- - - - - - - - T CF F CKQPGHWKADC - - - - - - - - - - - -- - - - T HE V I CHCCKGGGHHAREC - - - - - - - - - - - -- - - - T RDV L CYRCKGYGHVQRDC - - - - - - - - - - - -- - - - - - - - - CWT CGG - PHL AKSC - - - - - - - - - - - -- - - - - - - - - CF L CGG - PHMVREC - - - - - - - - - - - -- V L GE E T RT CF YCGKT GHL KREC - - - - - - - - - - - -- - - - - - - - E CYHCRERGHL RPNC - - - - - - - - - - - -- E T GVKPGSCF NCGE L GHF ADKC - - - - - - - - - - - -KS KF L KGV F CHGCGKEGHL KNVC - - - - - - - - - - - -- - - - - - - - - CHNCGKQGHL KKDC - - - - - - - - - - - -- - - - KHQF KCYNCGKSGHMARQC - - - - - - - - - - - -- - - - RAP L T CY SCGKPGH I ARDC - - - - - - - - - - - -NDNWKKDKQCF NCNQKGHL AY EC - - - - - - - - - - - -- - - - P KSGKCF KCGKT GY I AKNC - - - - - - - - - - - -- - - - - - - - - CYNCGKKGHYEREC - - - - - - - - - - - -- - - - - - - - L CYRCGSQEHF VAKC - - - - - - - - - - - -- - - - - - - - E CF HCGSP EHMVRNC - - - - - - - - - - - -S KKKGE I P T CF YCNKKGHYAT NC - - - - - - - - - - - -NE KKE F I P T CF T CKKSGHT APNC - - - - - - - - - - - -P KT S KNV I T CYRCGAAGHMQYDC - - - - - - - - - - - -ADCKHKEM I CF NCGEEGH I GSQC - - - - - - - - - - - -- - - - - - - I KCF KCHGYGHYQAQC - - - - - - - - - - - -P P S SRE T RT CYGCGKPGH I ARDC - - - - - - - - - - - -- - - - L RDQL CL YCGNNGHF I QAC - - - - - - - - - - - -- - - - - KL DL CYRCGEPGHRAGAC - - - - - - - - - - - -- - - - - - - - L CF KCGGKWGHAHRC - - - - - - - - - - - -- - - - - - - - L CF KCDAHY F KGHVC - - - - - - - - - - - -- - - - - - - - L CY SCDSKY T KGHKC - - - - - - - - - - - -- - - - - - NV T CYRCGQPGHF SNQC - - - - - - - - - - - -- - - - - - - T KCF SCNQEGH I S S KC - - - - - - - - - - - -- - - L I RT GKCF QCREPGH I S RNC - - - - - - - - - - - -Y QRRRE T GACL RCGNSGHQVADC - - - - - - - - - - - -- - - - - - NEDCF KCGRRGHF SRAC - - - - - - - - - - - -- - - - - - - - V CS SCHE T GHL RRMC - - - - - - - - - - - -- - - - - - - KT CKKCGKQNHF AKCC - - - - - - - - - - - -- - - - - AL DF CWQCGR I GVRT VACC - - - - - - - - - - -- - - - - - - - ACF RCNEMGH I AWNC - - - - - - - - - - - -- - - - - - - S QCPHHPNSNHVAKDC - - - - - - - - - - - -P GYDANVRCDF HSGAPGHHT EKC - - - - - - - - - - - -- - - GGL DQYCDF HKRSGHS T AAC - - - - - - - - - - - -- - - - - - - - Y CD I HRV T RHL T KDC - - - - - - - - - - - -- - - - - - - RL CF YCKKEGHRL NEC - - - - - - - - - - - -- - - - - - R I I CYGCGT P E V YKPNC - - - - - - - - - - - -- - - - - - - - Y CL NCGKSNHS T S T C - - - - - - - - - - - -- - - - - - - - DEQYQVQDGEGNQL E E - - - - - - - - - - -- - - - - - Q I T CCE L CKGDHDT GF C - - - - - - - - - - - -KP SHSKG I QCHGCEGYGH I I AECP T HL KKHRKG - -- - - KRE PGSCYACGQL GHL VAQC - - - - - - - - - - - -RNSKRS E I QCHECQGYGH I KAECP S L KRKDL KCS EKRY VQF KF VCD I CQKS SHT T DMCWKK - - - - - - - - -KRYGNF P - P YQHCKKT NHL QKYCWWRPD - - - - - - -S RS RYKD I ECHYCGKKSH I KKY S - - - - - - - - - - - -S - NKF ANV ECHYCKKKGH I KRF C - - - - - - - - - - - -S KS RKE - I KCF YCGKPGH I KREC - - - - - - - - - - - -S KS RYKNV ECHYCHKT GH I KKNY - - - - - - - - - - - -NRSKSRVRNCYNCNQPGHF KRDC - - - - - - - - - - - -S KGAKPDDVCNYCKEKGHWKF DC - - - - - - - - - - - -L E KHS F KGKCY I CEKF GHRAREC - - - - - - - - - - - -S AKKRKDVVCYNCGERRHF KANC - - - - - - - - - - - -KHSKT T E I RCF KCNK I GHRVADC - - - - - - - - - - - -- QDAHS F L SCF KCGKT GHKA I QC - - - - - - - - - - - -KGNSKYKVKCHHCGREGH I KKDC - - - - - - - - - - - -RGGT DNRP T CQVCHKKGHVVADC - - - - - - - - - - - -T GGQDRRP T CQVCF KRGHT AADC - - - - - - - - - - - -V GGSEGRVKCQL CF KRGHT VMEC - - - - - - - - - - - -- GN I KKV L T CY T CGT I GHKS S ECQAKT KPK - - - - -E GL AY SD I T CYNCQQKGHKMRT CPAL NCKR - - - - -AF KKQYKGRCSNCGEYGHKSGDCSERDKP S SGT GNAF VKQF KGRCNKCGT YGHKAAT CP S VKNDGGDPGTL DKYGKRSRCA I CQS T F HWADC - - - - - - - - - - - - -L DRYGRRSKCA I CQS T F HWAKDC - - - - - - - - - - - -L DKL GRV SRCV I CDSKL HWAAKC - - - - - - - - - - - -L GT DGQV T RC I I CDSKF HWARGC - - - - - - - - - - - -NGKNKS E V T CF RCKKKGHKSY ECP - - - - - - - - - - -- - - - - KK I T CHRCRKPGHMKRDC - - - - - - - - - - - -- GKNKKD I QCF NCGKKGHYKSDCR - - - - - - - - - - -- L GGRNPGNCKNCGKRGHWAKDC - - - - - - - - - - - -- KS KPK I I T CYKCKQT GHYRNQC - - - - - - - - - - - -- - - - - - - - V CF YCKKPGHK I ADC - - - - - - - - - - - -- - - - - - N I KCHHCGKEGH I KRDC - - - - - - - - - - - -GGF S V P PDT CL YCKKT GHYKRKC - - - - - - - - - - - -- - - - - - - GGCF VCGSDQHWAREC - - - - - - - - - - - -- - - - - - KKECAKCHL L GHT AE EC - - - - - - - - - - - -- - P NKGRP I CS F YNRVGH I AERC - - - - - - - - - - - -RRNRRRS EGCF T CGERGHF AADCPNKRT F DQGNNS- - - - - - - - KCGYCGF T GHSENEC - - - - - - - - - - - -- - - - - - - - S CMYCKS V F HCS I NCK - - - - - - - - - - -- - - - - - L L T CDHCKKKGHT KDKCWL L HPHL KPAKF

Conservation

CxxCxxxxHxxxxC

Retroviridae

Ty3/Gypsy

Ty1/Copia

a

Supplementary Figure 5: GAG RNA binding domain conservation and EVD poly-

protein product production in EVD constructs.

(e) Alignment of GAG amino acid sequences from representative members of Retrovir-

idae and Pseudoviridae (Ty3/Gypsy and Ty1/Copia). The bottom sequence (black

arrow) corresponds to the EVD-encoded GAG protein. Coloured columns in the align-

ment indicate residues with conserved physico-chemical properties. The black box

displays the consensus sequence logo of highly conserved residues corresponding to

the RNA-binding motif (Cx2Cx

4Hx

4C). (c) Scheme of p35S:HA-EVD-FLAG and

p35S:HA-EVDǻ5%'-FLAG constructs, see methods for details. (d) Accumulation of the

EVD polyprotein N-terminal and C-terminal products from p35S:HA-EVD-FLAG and

p35S:HA-EVDǻ5%'-FLAG was assessed by Western analysis of total proteins extracted

from N. benthamiana-infiltrated leaves at 4 dpi. Equal loading was verified by Coomas-

sie staining of the membranes after blotting.



5’LTR 3’LTR GAG PR IN RT-RNase

a

5630000 5631000 632000 5633000 634000 5635000

100

010

0

Coordinates

Abun

danc

e of

siR

NAs

(rpm

) s

as

epi15 F14

21-22 nt siRNAs

0

5

10

15

20

25

35

30

epi454 F8 epi439 F8

b

EVD

cop

y nu

mbe

r


Supplementary Figure 6: Mapping of 21-22-nt siRNAs to EVD in epi15 F14 and

EVD copy number in epi454 and 438 F8.

(a) Deep sequencing analysis of 21-22-nt siRNAs mapping to EVD in epi15 F14. y

axis: number of normalized small RNA reads per million sequences (rpm); x axis:

Arabidopsis chromosome 5 coordinates corresponding to the EVD locus found in

Col-0 (TAIR 9). s, sense; as, antisense. (b) EVD copy number in epi454 and 439 F8

calculated as the total EVD insertions inferred from the number of heterozygous and

homozygous insertions obtained by sequencing (Gilly et al, submitted) including the

original EVD and ATR loci.


EVD (AT5G17125��·LTR

WT

epi1

5 F8

ep

i15

F11

epi1

5 F1

4

a b

c

d


ATR

EVD

EVD insertions in epi439 F8

20

30

40

50

10

0

EVD

cop

y nu

mbe

r

ND

epi439 F9 1 2 3

epi439 F15 epi439 F17

40%

60%

80%

100%

20%

0%

epi439 F9 epi439 F15 epi439 F17

% D

NA

met

hyla

tion

ND

Heterozygous

Ch.I

Ch.II

Ch.III

Ch.IV

Ch.V

1 2 3 1 2 3 4

1 2 3 1 2 3 1 2 3 4

Supplementary Figure 7: Bisulfite sequencing dot-plots of EVD LTR in epi15

generations and EVD copy number and DNA methylation in epi439 generations.

(a) Dot-plots depiction of bisulfite sequencing data obtained for individual clones used

to generate the results shown in Figure 4a and Supplementary Table 2. Filled circles

indicate methylated cytosine and empty circles unmethylated cytosine. (b) Scheme of

Arabidopsis chromosomes showing EVD insertions found in the ddm1-derived epi439

line at F8. The original EVD and ATR loci are marked with blue triangles, red triangles

indicate new transposition events. The three new insertions were selected for further

analysis. (c) EVD copy number measured by qPCR in individual plants of epi439 F17.

EVD copy number was not determined (ND) in the F9 and F15 generations. Error bars

represent standard deviation from two independent experiments involving duplicate

PCRs each. (d) McrBC-qPCR-based methylation analysis at the LTRs of EVD new

insertions shown in (b) in the epi439 F9, -F15 and -F17 individual plants used in (c).

Error bars represent standard deviation from duplicate PCRs in c-d.

CHH CHG CG


0

10

20

30

40

50

05

101520253035404550

WT WT x epi15 F14 epi15 F14

EVD

cop

y nu

mbe

r

EVD

cop

y nu

mbe

r

@LTR

#�·*$*

@U6

-21nt -24nt

-21nt -24nt

}F1�

F2

a(1)

(2)

(3)

b

21-to

-24-

nt C

orre

latio

n


Supplementary Figure 8: Correlation of LTR 24-nt siRNAs abundance with EVD copy number and

21-24-nt siRNAs mapping correlation on EVD.

(a) To explain the PTGS-to-TGS shift in EVD silencing, we considered the possible emergence of discrete

EVD loci with peculiar features allowing production of 24-nt LTR siRNAs with potent trans-TGS activities.

This hypothesis had two predictable and interrelated implications: (i) such loci would produce dispropor-

tionate amounts of 24nt siRNAs independently of EVD copy numbers and, (ii) these loci would be easily

segregated away from an F14. We therefore tested these ideas here. (1) EVD copy number measured by

qPCR in WT, epi15 F14 and the F1 progeny of the F14 X WT cross. (2) Same as in (1) but in distinct

plants of the F2 progeny, grouped according to their respective copy numbers. Error bars represent

standard deviation from two independent experiments involving triplicate PCRs each. (3) Northern analy-

sis of EVD LTR 24-nt siRNA levels in F2 progeny plants containing similar numbers of EVD insertions as

determined in (2). U6 detection is shown as loading control. The results clearly show that (i) all F2s, with

no exception, produce 24nt siRNAs and (ii) 24nt-LTR siRNA levels strictly correlate with the copy number

of segregating EVD copies, making the emergence of the hypothesized, specific EVD loci highly unlikely.

(b) Pearson correlation coefficient (Y-axis) between the 5’-end of all 24mers and all 21-mers mapping the

2,000 first nucleotides of EVD with the computation of virtual a lag from -10 to +10 nucleotides (X-axis)

between the first nucleotide of all 21-mers and all 24-mers. This allows to assess if DCLs process the

24-mers and the 21-mers at the same position of a shared dsRNA substrate.

0.00

0.05

0.10

0.15

0.20

0.25

0.30

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10

Lag (nt)


WT

epi1

5 F8

ep

i15

F11

epi1

5 F1

4 ep

i15

F15

RPP4 (AT4G16860) SOLO LTRSupplementary Figure 9: Bisulfite sequencing

dot-plots of RPP4 Solo-LTR in epi15 generations.

Dot-plots of bisulfite sequencing from individual clones for

results shown in Figure 6b and Supplementary Table 4.

Filled circles indicate methylated cytosine and empty

circles unmethylated cytosine.

CHH CHG CG



M

M

M

p p

M

M

M

}

M

�·LTR �·LTR GAG PR IN RT-RNase

GAG PR IN RT

RDR6

DCL4/2

AGO1/2


GAG PR IN RT

RDR6

DCL4/2


DCL3

AGO4

RDR2DCL3

AGO4

PolV/IV

AGO1/2

PolV/IV

?

FM

FM

ov ov

VLP VLP

ORF 21-22-ntsiRNAs

ORF 21-22-ntsiRNAs

ORF 24-ntsiRNAs

LTR 24-ntsiRNAs

Reve

rse

Tran

scrip

tion

/ Int

egra

tion

Reve

rse

Tran

scrip

tion

/ Int

egra

tion

DNA methylationDNA methylation


DNA methylation

SOLO LTR

DNA methylation

cis-

trans-� �

i) ii) iii)

(1)

(2) (3)

(4)

(5) (6)

(7)(8)

(9)

(10)

(10)

Supplementary Figure 10a

b


Supplentary Figure 10: EVD life cycle in Arabidopsis.

(a) EVD expression pattern ensures host integrity through developnet and transmis-

sion of new insertions to the next generation. Once reactivated, adaxial L2-specific

EVD expression (in red) restricts potential transposition to somatic tissue. Meristem

(harbouring the stem cells from which aerial organs derive; M) exclussion will then

allow the plant to develop without the accumulation of EVD new insertions. However,

L2 expression and transposition in gynoecia (flower female reproductive organ),

particularly at the placental regions (P) which give rise to the ovules (ov), from which a

L2 cell differentiates into the haploid female megaspore (FM) , will allow transmission

of EVD new insertions (see Supplementary Fig.2 for more details). Therefore, after

fertilization, and embryo development, each seed will display different number and

pattern of EVD insertions. (b) Rise and demise of EVD. In early proliferation stages

(A), a low EVD copy number (light blue; i) accounts for moderate transcription and

dsRNA synthesis by RDR6 (1). Processing of dsRNA by DCL4 and DCL2 produces

3’GAG 21-22-nt siRNAs (2) loaded into AGO1/2 (3) that can only partly degrade the

EVD RNA (4) due to its protection by GAG as part of putative viral-like particles (VLPs;

5). Because single seeds acquire distinct patterns and numbers of EVD insertion over

generations, an increasing amount of progeny plants will attain the ֤40 copies thresh-

old (dark blue; ii) beyond which saturating dsRNA levels allow processing, by DCL3, of

3’GAG 24-nt siRNAs loaded into AGO4 (6), these induce de novo methylation of the

3’GAG DNA (7), which, in turn, initiates antisense transcription, possibly by PolIV/PolV,

and coordinated spreading of methylation towards the LTR (8). Eventual epigenetic

silencing of EVD is achieved over a few subsequent generations (red, iii) resulting in

TGS maintained at the LTR by RdDM (9). Ensuing 24-nt LTR siRNAs display potent

cis- and trans-silencing properties (10).


Total % methylated # Clones # Exp

Wilson Score Interval (95% confidence level)

CG CHG CHH CG CHG CHH CG int- CHG int- CHH int- CG int+ CHG int+ CHH int+

EVD

WT 85 102 1274 84,70% 7,84% 10,12% 17 2 75,57% 4,03% 8,58% 90,83% 14,71% 11,90% met1-3 80 96 1199 6,25% 3,12% 3,00% 14 2 2,70% 1,07% 2,17% 13,81% 8,78% 4,13%

epi15 110 132 1647 3,66% 1,51% 1,94% 22 2 1,44% 0,41% 1,38% 9,01% 5,35% 2,73%

ATR

WT 80 96 1197 88,75% 18,75% 22,05% 16 2 79,98% 12,20% 19,79% 93,97% 27,70% 24,49% met1-3 103 125 1560 3,88% 4,00% 6,28% 21 2 1,52% 1,72% 5,18% 9,56% 9,02% 7,59%

epi15 89 106 1345 85,39% 17,92% 21,04% 18 2 76,59% 11,78% 18,95% 91,26% 26,30% 23,30%

Supplementary Table 1

Supplementary Table 1: Figure 1 and Supplementary Figure 1 bisulfite sequencing raw

data.

Raw data from the bisulfite sequencing analysis in Figure 1b and Supplentary Figure 1. The

number of CG, CHG and CHH sites as well as the number of sequenced clones are indicated.

Wilson score intervals are shown for a confidence level of 0.95.


Total % methylated # Clones # Exp

Wilson Score Interval (95% confidence level )

CG CHG CHH CG CHG CHH CG int - CHG int - CHH int - CG int + CHG int + CHH int +

WT 90 108 1350 82,22% 10,28% 3,92% 18 2 73,05% 5,85% 3,01% 88,75% 17,44% 5,09% epi15 F8 75 90 1125 2,66% 1,12% 2,76% 15 2 0,73% 0,20% 1,95% 9,20% 6,04% 3,89%

epi15 F11 75 90 1125 8,00% 3,33% 3,82% 15 2 3,72% 1,14% 2,85% 16,37% 9,34% 5,11% epi15 F14 90 108 1350 93,33% 21,29% 13,85% 18 2 86,21% 14,63% 12,11% 96,91% 29,93% 15,80%



data.

Raw data from the bisulfite sequencing analysis in Figure 4a and Supplementary Figure 7. The




CG CHG CHH CG CHG CHH CG int- CHG int- CHH int- CG int+ CHG int+ CHH int+WT 176 80 581 94,88% 32,50% 8,60% 16 2 90,56% 23,24% 6,58% 97,28% 43,36% 11,16%epi15 F8 182 89 660 4,39% 3,37% 2,57% 18 2 2,24% 1,15% 1,61% 8,43% 9,45% 4,08%epi15 F11 194 90 663 26,28% 4,44% 2,26% 18 2 20,59% 1,74% 1,37% 32,89% 10,87% 3,70%epi15 F14 252 115 850 60,31% 33,91% 6,58% 23 2 54,16% 25,90% 5,10% 66,15% 42,96% 8,45%WT 117 104 585 77,77% 2,88% 0,68% 13 2 69,42% 0,98% 0,26% 84,35% 8,13% 1,74%epi15 F8 162 144 810 5,55% 0,69% 0,86% 18 2 2,95% 0,12% 0,42% 10,21% 3,82% 1,77%epi15 F11 198 176 990 30,80% 11,36% 5,65% 22 2 24,79% 7,47% 4,38% 37,54% 16,90% 7,27%epi15 F14 180 159 899 77,33% 8,80% 3,67% 20 2 70,68% 5,31% 2,62% 82,84% 14,23% 5,11%

Total % methylated Wilson Score Interval# Clones # Exp

5'GAG

3'GAG


Supplementary Table 3: Figure 5 bisulfite sequencing raw data.

Raw data from the bisulfite sequencing analysis in Figure 5d-g. The number of CG, CHG and

CHH sites as well as the number of sequenced clones are indicated. Wilson score intervals are

shown for a confidence level of 0.95.


CG CHG CHH CG CHG CHH CG int- CHG int- CHH int- CG int+ CHG int+ CHH int+WT 189 112 1451 0,00% 0,89% 1,72% 19 2 0,00% 0,16% 1,17% 1,99% 4,88% 2,53%epi15 F8 180 108 1385 2,22% 0,00% 1,01% 18 2 0,87% 0,00% 0,60% 5,57% 3,43% 1,69%epi15 F11 190 113 1460 1,05% 0,00% 1,43% 19 2 0,29% 0,00% 0,94% 3,75% 3,29% 2,18%epi15 F14 189 114 1460 13,75% 10,52% 17,39% 19 2 9,56% 6,12% 15,53% 19,39% 17,49% 19,42%epi15 F15 200 119 1533 51,11% 26,05% 25,11% 20 2 44,23% 19,00% 23,00% 57,95% 34,60% 27,34%

Total % methylated Wilson Score Interval (95% confidence level)# Clones # Exp



data.

Raw data from the bisulfite sequencing analysis in Figure 1b and Supplementary Figure 9. The




Supplementary Table 5: List of primers.(Non%converted,bisulfite,primers,sequence,is,indicated,in,braquets)

EXPERIMENT PRIMER SEQUENCE (5'-3')EVD-RT F GATCTTGGTGAACTCAAATACTTEVD-RT R TACTCCCCCTCAAGCGCAAGACEVD-qRT F GATAGAGGAGATAGAAGATCTACAACTGGEVD-qRT R CTCTATACTCCGATTCTGCACTCGAACAEVD-P F TTTCATGGTATCAGAGCATAAGATEVD-P R CACCCGAGTCTATCACAATTGAAC

EVD-LTR F TTGATCAAGACTCAAATAAGAAAGGCCEVD-LTR R TATGCTCTGATACCATGAAAGAATATEVD-P F TTTCATGGTATCAGAGCATAAGATEVD-5G R TTTTTAAGTGTATCCCACAACTCTTEVD-3G F TAAGTCAAGAAGACTTAGAGTTTAEVD-3G R ACTTTGCTCCTCATGAGTTTCTTEVD-PI F GGTGGTTCAAAAGGAGAACTCTATGTTCTEVD-PI R GAAACATCATTGACCGCGCCACCTCCATEVD-IR F GAAGGATTGGGAGAACCTAAAAGATCTTACEVD-IR R CTTATACTTGATTGTGAATACCCACCTTGAEVD-RR F CAAAGACGGTATAGACTCTACCAAGACEVD-RR R CTCTATACTCCGATTCTGCACTCGAACAEVD-RN F GATAGAGGAGATAGAAGATCTACAACTGGEVD-RN R TACTCCCCCTCAAGCGCAAGACmGFP-F AGTAAAGGAGAAGAACTTTTCACTmGFP-R TTCCGTCCTCCTTGAAATCGAEVD ORF attB1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGAGACTTCTCAAAAGATGEVD ORF attB2 GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAAGAGTGAGATAGATCCEVD dRBD F AGGATAGAAAACTGTTGACTTGGTTGCTACATCCACACCTEVD dRBD R AGGTGTGGATGTAGCAACCAAGTCAACAGTTTTCTATCCTHA-EVD F ATGTACCCATACGATGTTCCAGATTACGCTGGTGGAGGCGAGACTTCTCAAAAGATGAEVD-FLAG R TCACTTGTCATCGTCATCCTTGTAATCGCCACCTCCAGAGTGAGATAGATCCACAAHA-EVD attB1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTACCCATACGATGTTCCEVD-FLAG attB2 GGGGACCACTTTGTACAAGAAAGCTGGGTTTCACTTGTCATCGTCATCCTTGT454 EVD-A F CTACTGGCGTGGATGCTTTT454 EVD-A R GTAAACAGAGTTCAGATTTAAACCAA454 EVD-B F TTCTTCCAAAATTTGATTACTTGC454 EVD-B R TCCGAAAATGGCATAAGAGG454 EVD-C F TTGGAATTCGATTCAACTAGAGG454 EVD-C R CTCAAATTTCATTGGAGACACATC454 EVD-D F TGTGCGTAGCTTTTCCTTGA454 EVD-D R ATTGAGGAACCAGCATCACC454 EVD-E F GCCTTAATGAATGGCCCAATA454 EVD-E R ACTCCACGGGACTACCTCAC454 EVD-F F GACCCCAACCAATCAATCAC454 EVD-F R GACGTTATGGTGGAATATGTGC454 EVD-G F AAAATGTCACTTATCATTGCAAGC454 EVD-G R TTTTAGTGGTGCGGGCTTTA454 EVD-H F CCAACGTGGGTTAGATCCAT454 EVD-H R ACGGCTATTGGAGTTTTTGA454 EVD-I F CGGTGTTTGTAGTGGGAACC454 EVD-I R GCAAAAACATTCGTACATTTCG454 EVD-J F TCTCTTGTCTTGTGCGACTCA454 EVD-J R CACACCATGGATAATGCTCTT439 EVD-ChI F ATCAACAAATAGATATCTTTGCATTTT439 EVD-ChI R TTCTTTTCGGATTCTGTGTGG439 EVD-ChIII F ATGGTTCGATTGTGGAGGAA

EVD-LTR BS F TTTATAARCATAAAAACATAATCTTATRCTCTAATACCATA (TTTATAAGCATAAAGACATGATCTTATGCTCTGATACCATG)

EVD-LTR BS R GGAAAAAAAYGAAGGAATTTTAGAATAYGATTTGGTAATTT (GGAAAAAAACGAAGGAACCCTAGAACACGATTTGGCAATTC)

ATR-LTR BS R TTTTAATGAGGTGATGAATTATGTGTTTTAAGTTTAGATTT (TTTTAATGAGGTGATGAATCATGTGTTCCAAGCTTAGACTC)

RT-RN probe

EVD RT-PCR/RFLP

EVD copy# qPCR/qRT-PCR

EVD ISH/Southern/ HMW-Northern probe

LTR Bisulfite PCR

LTR probe

5'GAG probe

3'GAG probe

PR-IN probe

IN-RT probe

RN probe

mGFP probe

EVD WT GAG-POL ORF CloningEVD dRBD

Site-directed Mut.

EVDHA-GAG-POL-FLAG

Cloning

epi454/439 EVDinsertionszygosity


439 EVD-ChIII R CAACATTCTAGGGTTTTACCTTGC439 EVD-ChV F TCTCCCACCCAAAATCTCAG439 EVD-ChV R GCGTGTTGTTTTGCAGTGATEVD-LB R ACTGGTCATTGGTTGACCTTGEVD-RB F CCAAGACCAAGGAACCTCTTC454 EVD-A qF ATGAAAATATTTGGCGTGTTCAT454 EVD-A qR TCGAATCTGTCTTCTAGATTATGTGTG454 EVD-B qF GCTCAGAGGCAGTTGTGAAAG454 EVD-B qR ACTTTTCAAGCAACGTTGTGC454 EVD-C qF CATCAGCTTGATGATCTACAGAGAG454 EVD-D qF TTGTCCCGAGGATAGTCTTC454 EVD-D qR ACCAGAGTATATAGAACATGATATTGAAGTG454 EVD-E qF CGTTTTAAGACGAAGGGTGTG454 EVD-E qR AGGACTGACGTCACACCCTA454 EVD-F qF CCTTTCGAGTCGAGATCCAAT454 EVD-F qR TCATATTCACATATGTTGGTGTTGG454 EVD-G qF CTGTACATTTCAATATTTTATCCATCTTG454 EVD-G qR AAATCACAACATATGTAAATGTATCAAGT454 EVD-H qF GAAGACTTTATGCCGTTCATCA454 EVD-H qR TTACATTCTTAGCGTTTCCTGGT454 EVD-I qF TTCAGTTTCATTAATAAGAACCAGGTTAG454 EVD-J qF AAAACCTGGTACCTTCTCTGTGA454 EVD-J qR TCAAAAACATTCAGAAGGTCAGAG439 EVD-ChI qF AAAGCAGTCGAGAATAAGAACCA439 EVD-ChI qR AGATCTTCTGTTTTGATGCAAGTC439 EVD-ChIII qF TGAGGAGTTGAATTCTGTGATGA439 EVD-ChIII qR CTTGAGCCCCAAGGAAGTC439 EVD-ChV qF CCAGTTTGACGAAATTAAAACAA439 EVD-ChV qR CGTAACCAAACATAAGGAGAACC

RPP4-qRT F ACATGCGAGGACAAACCAGAGGRPP4-qRT R TTCAATTCCGACGAAGTCACC

AT1G68420 qF GTCTCACACTTCAAGGCATCTTGCAT1G68420 qR TTCATAGCTTCCTCTACACCGGCAAT5G25550 qF GTTACAAGCGTGGAAACGTGCCATAT5G25550 qR TGTAGTCGCACACACGTGATCCAAACT2 qF GCACCCTGTTCTTCTTACCGACT2 qR AACCCTCGTAGATTGGCACAAT5G13440 qF ACAAGCCAATTTTTGCTGAGC AT5G13440 qR ACAACAGTCCGAGTGTCATGGTAT2G36060 qF TGAAGTCGTGAGACAGCGTTGAT2G36060 qR GGGCTTCTCCATTGTTGGTCAT4G29130 qF GGCGTTTTCTGATAGCGAAAAAT4G29130 qR ATGGATCAGGCATTGGAGCT

EVD-5GAG BS F ATTTTAAGGGGGAAATTATTTAATTTGGTTAAGAATAAT(ACTTCAAGGGGGAAACTACCTAACTTGGTCAAGAACAAC)

EVD-5GAG BS F ACCTAAACTTACCAAAATATTTAATAAACTCTAAATCTTCTTA(ACCTGAACTTACCAAAGTGTTTAGTAAACTCTAAGTCTTCTTG)

RPP4 qRT-PCR

5'GAGBisulfite PCR

3'GAGBisulfite PCR

EVD-3GAG BS F GTGTTTGAAGTGGAAGAAGGYGATTAAYGAATTAA (GTGTTTGAAGTGAAGAAGGCGATTAACGAACTAA)

EVD-3GAG BS F ATAACCCRACTTAACTTTRCTCCTCATAAATTTCTTAAA(ATGACCCGGCTTGACTTTGCTCCTCATGAGTTTCTTGAG)

AT1G68420qRT-PCR

RPP4-SLTR BS F GGGAGGYGTTTAGGTAATTTATAYGTATATTATATATTGATT (GGGAAGGCGTCCAGGTAATTCATACGTATATTATATATTGATC)

RPP4-SLTR BS R TTCCTCTACAAAAATATTTTAAAAACACAAATTTTATAATTATCAA(TTCCTCTACAAAGATATTTTGAAAACACAAATTTTGTGGTTATCAG)

AT5G25550qRT-PCR

qRT-PCRControls

epi454/439 EVDinsertionszygosity

epi454/439 EVDinsertion

specific McRB-qPCRmethylation analysis

RPP4 SOLO LTRBisulfite PCR


supplementary information · expressing evd: epi12 (f9) and epi35 (f9) previously described in...

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