genomic dna and total rna were isolated from inflorescence tissue collected from columbia-0 wildtype ( wt ) and loss of function lines in ddm1 - 2 , rdr6 - 15 , ddm1 - 2 rdr6 - 15 , and ddm1 - 2 dcl1 - 11 in a columbia-0 background , grown under standard long - day growth conditions . the ddm1 - 2 line used in this study was in the fifth generation of inbreeding and used for the genetic cross rdr6 - 15 . double heterozygous lines were selected , selfed and second generation ddm1 - 2 rdr6 - 15 were isolated for sequencing in this study . dna was isolated from pooled inflorescence tissue ( n = 3 ) collected phenol / chloroform extraction . total rna was isolated from pooled inflorescence tissue ( n = 3 ) and trizol ( invitrogen ) extraction following manufacturers instructions . pooled inflorescence tissue between stage 9 and 11 ( unopened / just opened flowers ) ( n = 3 ) . col-0 , ddm1 - 2 , rdr6 - 15 and ddm1 - 2 rdr6 - 15 truseq small rna sequencing libraries , sequenced on illumina hiseq . for the col-0 , ddm1 - 2 replicates , and ddm1 - 2 dcl1 - 11 small rna sequencing libraries were prepared by nebnext multiplex small rna libraries , sequenced on illumina miseq . mapping srna - seq was performed using bowtie from the open - source tuxedo suite , allowing for both unique ( u ) and multiple ( m ) mapping reads , normalized by reads per million ( rpm ) . for each library , 15 g of genomic dna was sheared using a covaris s220 adaptive focused acoustics ultra sonicator . libraries were constructed following standard protocol using the neb next dna sample prep master mix set 1 ( neb e6040 ) and illumina - compatible paired - end adaptors , which had all cytosines , methylated . 50 ng of each library was treated with sodium bisulphite using ez dna methylation - gold kit ( zymo research d5005 ) according to the protocol provided by the manufacturer . for each purified library 510 ng of purified library was amplified using expand high fidelity plus pcr system ( roche 03300242001 ) , which is capable of efficiently amplifying uracil - containing templates . 50 l reactions contained 200 m each dntp , 1 m primer , 2.5 mm mgcl2 and 2.5 u expand hifi enzyme , and were performed according to the manufacturer s instructions for 18 cycles . fragments of 220350 bp were excised from gel and purified using the qiaquick pcr purification kit ( qiagen 28104 ) . dna concentrations were quantified on bioanalyzer ( agilent ) , diluted to 10 nm and loaded on flow cells to generate clusters . libraries were sequenced on illumina gaii or hiseq2000 machines using the paired - end 50 cycles protocol . library read count given in supplementary table 8 . for statistical analysis of te methylation in ddm1 rdr6 replicates compared to ddm1 , we performed binomial exact test , alternative = two - sided , taking the total methylation per transposon superfamily and asking the number of tes that are more methylated in ddm1 rdr6 compared to ddm1 , and the number of tes less methylated , p - value < 2.2e-16 alternative hypothesis : true probability of success is not equal to 0.5 , 95 % confidence interval , sample estimates : probability of success given ( supplementary table 10 ) . 128 precursors were identified , representing 96 unique mirna candidates . among these 96 sequences , t / r / sn / snorna related reads were filtered and the mirna abundance to be at least 10 ( normalized abundance ) in one or more libraries and with genome hits no more than 20 . the srnas passed this filter were mapped to the genome and precursor structure was checked by mireap , developed by bgi , http://sourceforge.net/projects/mireap/ ) . each precursor was defined by extending from the abundant tag ( as potential mirna ) on either side , up to 200-nt to the potential mirna - star site . after this , two additional filters ( strand bias and top1 + top2 ratio ) were applied to distinguish mirna from sirna loci . strand bias was calculated by the sum of srna abundance on sense strand divided by the total abundance on both strands . top1 + top2 is the proportion of the abundance of top2 abundant tags , also referred as distribution filter . the number of known ath - mirnas remaining served as an indicator of the efficiency of the filtering . centroidfold was used with default settings to visualize the overall mirna precursor structure for manual evaluation . mirna target prediction was performed using the noble srna targeting resource http://plantgrn.noble.org / psrnatarget/. mrna purification using invitrogen dynabeads mrna purification kit . 5 adaptor ligation . reverses transcription for pare following first strand cdna synthesis , ( short pcr ) 7 cycles , ( long pcr ) 35 cycles . pcr amplification of pare library ( long pcr ) 15 cycles followed by page purification . 5-pare rna adaptor 5-guucagaguucuacaguccgac-3. target rt - primer : 5 cga gca cag aat taa tac gac ttt ttt ttt ttt ttt ttt . short pcr primer : 5-adapter primer 5-gttcagagttctacagtccgac-3 3-adapter primer 5-cgagcacagaattaatacgact-3 dsdna adapter ( pare truseq duplex ) ( page purified ) : dsdna_2_top : 5-tgg aat tct cgg gtg cca agg dsdna_2_bottom : ( 5phos ) 5-cct tgg cac ccg aga att cca nn final pcr primer ( p2 srna long primer ) 5 : aatgatacggcgaccaccgacaggttcagagttctacagtccga . pare - sequencing analysis performed by defining two flanking windows at each predicted target site . the sum of abundance of pare tags matching to ( 1 ) a small window ( ws ) of 5-nt ( cleavage site  2-nt ) , and ( 2 ) a long window ( wl ) of 31-nt ( cleavage site 15 nt ) was calculated . cleavage sites were filtered to retain those for which ws / wl  0.5 in the pare - seq libraries ; sites failing this criteria were considered background mrna cleavage levels . some chloroplast contamination can account for the distribution of raw read count abundances in our libraries . however , when comparing to the normalization basis , and the mapping of pare - tags to multiple loci , these reads appear to be of chloroplast mrna in origin . the mirna target lists were filtered by combining both mirna target penalty score (  7 ) with pare - seq data filters of ws / wl  0.3 and ws  3 ( strict ) , or relaxed ; mirna target penalty score (  7 ) with pare - seq data filters of ws / w  0.3 and ws  2 . we performed the chi - square goodness of fit test , calculating the p - value for observed value given the expected value . if we assume pare tags fall randomly on all the positions in the long window ( 31-nt ) , the expected frequency for pare reads fall into the small window ( 5-nt ) would be 5/31 , therefore , the expected frequency would be would 26/31 for other regions . atgrf8 , target of mir396 , in col-0 , we observe 603 in the small window , and , 614 in the long window ( 614  603 = 11 in other region ) , the chi - squared test for given probabilities c(603 , 11 ) x - squared = 3057.857 , df = 1 , p - value < 2.2e-16 ( supplementary table 3 ) . for specific mirna targeting , the sum of abundance of pare tags matching to ( 1 ) a shorter small window ( wss ) of 1-nt ( cleavage site 0-nt ) , and ( 2 ) a shorter long window ( wsl ) of 3-nt ( cleavage site 1nt ) was calculated ( supplementary table 9 ) . as per instructions 5 rlm - race adapter ligation followed by rt - pcr , followed by two pcrs ( outer and inner ) , cloning and sequencing . 5 rlm - race adapter : 5-gcugauggcgaugaaugaacacugcguuugcuggcuuugaugaaa-3. 5 rlm - race outer primer : 5-gctgatggcgatgaatgaacactg-3. 5 rlm- race inner primer : 5-cgcggatccgaacactgcgtttgctggctttgatg-3. genomic dna and total rna were isolated from inflorescence tissue collected from columbia-0 wildtype ( wt ) and loss of function lines in ddm1 - 2 , rdr6 - 15 , ddm1 - 2 rdr6 - 15 , and ddm1 - 2 dcl1 - 11 in a columbia-0 background , grown under standard long - day growth conditions . the ddm1 - 2 line used in this study was in the fifth generation of inbreeding and used for the genetic cross rdr6 - 15 . double heterozygous lines were selected , selfed and second generation ddm1 - 2 rdr6 - 15 were isolated for sequencing in this study . dna was isolated from pooled inflorescence tissue ( n = 3 ) collected phenol / chloroform extraction . total rna was isolated from pooled inflorescence tissue ( n = 3 ) and trizol ( invitrogen ) extraction following manufacturers instructions . pooled inflorescence tissue between stage 9 and 11 ( unopened / just opened flowers ) ( n = 3 ) . col-0 , ddm1 - 2 , rdr6 - 15 and ddm1 - 2 rdr6 - 15 truseq small rna sequencing libraries , sequenced on illumina hiseq . for the col-0 , ddm1 - 2 replicates , and ddm1 - 2 dcl1 - 11 small rna sequencing libraries were prepared by nebnext multiplex small rna libraries , sequenced on illumina miseq . mapping srna - seq was performed using bowtie from the open - source tuxedo suite , allowing for both unique ( u ) and multiple ( m ) mapping reads , normalized by reads per million ( rpm ) . library construction and bisulphite conversion were carried out , essentially , as described in . for each library , 15 g of genomic dna was sheared using a covaris s220 adaptive focused acoustics ultra sonicator . libraries were constructed following standard protocol using the neb next dna sample prep master mix set 1 ( neb e6040 ) and illumina - compatible paired - end adaptors , which had all cytosines , methylated . 50 ng of each library was treated with sodium bisulphite using ez dna methylation - gold kit ( zymo research d5005 ) according to the protocol provided by the manufacturer . for each purified library 510 ng of purified library was amplified using expand high fidelity plus pcr system ( roche 03300242001 ) , which is capable of efficiently amplifying uracil - containing templates . 50 l reactions contained 200 m each dntp , 1 m primer , 2.5 mm mgcl2 and 2.5 u expand hifi enzyme , and were performed according to the manufacturer s instructions for 18 cycles . fragments of 220350 bp were excised from gel and purified using the qiaquick pcr purification kit ( qiagen 28104 ) . dna concentrations were quantified on bioanalyzer ( agilent ) , diluted to 10 nm and loaded on flow cells to generate clusters . libraries were sequenced on illumina gaii or hiseq2000 machines using the paired - end 50 cycles protocol . library read count given in supplementary table 8 . for statistical analysis of te methylation in ddm1 rdr6 replicates compared to ddm1 , we performed binomial exact test , alternative = two - sided , taking the total methylation per transposon superfamily and asking the number of tes that are more methylated in ddm1 rdr6 compared to ddm1 , and the number of tes less methylated , p - value < 2.2e-16 alternative hypothesis : true probability of success is not equal to 0.5 , 95 % confidence interval , sample estimates : probability of success given ( supplementary table 10 ) . 128 precursors were identified , representing 96 unique mirna candidates . among these 96 sequences , t / r / sn / snorna related reads were filtered and the mirna abundance to be at least 10 ( normalized abundance ) in one or more libraries and with genome hits no more than 20 . the srnas passed this filter were mapped to the genome and precursor structure was checked by mireap , developed by bgi , http://sourceforge.net/projects/mireap/ ) . each precursor was defined by extending from the abundant tag ( as potential mirna ) on either side , up to 200-nt to the potential mirna - star site . after this , two additional filters ( strand bias and top1 + top2 ratio ) were applied to distinguish mirna from sirna loci . strand bias was calculated by the sum of srna abundance on sense strand divided by the total abundance on both strands . top1 + top2 is the proportion of the abundance of top2 abundant tags , also referred as distribution filter . the number of known ath - mirnas remaining served as an indicator of the efficiency of the filtering . centroidfold was used with default settings to visualize the overall mirna precursor structure for manual evaluation . 5 adaptor ligation . reverses transcription for pare following first strand cdna synthesis , ( short pcr ) 7 cycles , ( long pcr ) 35 cycles . pcr amplification of pare library ( long pcr ) 15 cycles followed by page purification . 5-pare rna adaptor 5-guucagaguucuacaguccgac-3. target rt - primer : 5 cga gca cag aat taa tac gac ttt ttt ttt ttt ttt ttt . short pcr primer : 5-adapter primer 5-gttcagagttctacagtccgac-3 3-adapter primer 5-cgagcacagaattaatacgact-3 dsdna adapter ( pare truseq duplex ) ( page purified ) : dsdna_2_top : 5-tgg aat tct cgg gtg cca agg dsdna_2_bottom : ( 5phos ) 5-cct tgg cac ccg aga att cca nn final pcr primer ( p2 srna long primer ) 5 : aatgatacggcgaccaccgacaggttcagagttctacagtccga . pare - sequencing analysis performed by defining two flanking windows at each predicted target site . the sum of abundance of pare tags matching to ( 1 ) a small window ( ws ) of 5-nt ( cleavage site  2-nt ) , and ( 2 ) a long window ( wl ) of 31-nt ( cleavage site 15 nt ) was calculated . cleavage sites were filtered to retain those for which ws / wl  0.5 in the pare - seq libraries ; sites failing this criteria were considered background mrna cleavage levels . some chloroplast contamination can account for the distribution of raw read count abundances in our libraries . however , when comparing to the normalization basis , and the mapping of pare - tags to multiple loci , these reads appear to be of chloroplast mrna in origin . the mirna target lists were filtered by combining both mirna target penalty score (  7 ) with pare - seq data filters of ws / wl  0.3 and ws  3 ( strict ) , or relaxed ; mirna target penalty score (  7 ) with pare - seq data filters of ws / w  0.3 and ws  2 . we performed the chi - square goodness of fit test , calculating the p - value for observed value given the expected value . if we assume pare tags fall randomly on all the positions in the long window ( 31-nt ) , the expected frequency for pare reads fall into the small window ( 5-nt ) would be 5/31 , therefore , the expected frequency would be would 26/31 for other regions . atgrf8 , target of mir396 , in col-0 , we observe 603 in the small window , and , 614 in the long window ( 614  603 = 11 in other region ) , the chi - squared test for given probabilities c(603 , 11 ) x - squared = 3057.857 , df = 1 , p - value < 2.2e-16 ( supplementary table 3 ) . for specific mirna targeting , the sum of abundance of pare tags matching to ( 1 ) a shorter small window ( wss ) of 1-nt ( cleavage site  0-nt ) , and ( 2 ) a shorter long window ( wsl ) of 3-nt ( cleavage site 1nt ) was calculated ( supplementary table 9 ) . as per instructions 5 rlm - race mapping the cleavage of mirna firstchoice ( ambion ) . 5 rlm - race adapter ligation followed by rt - pcr , followed by two pcrs ( outer and inner ) , cloning and sequencing . 5 rlm - race adapter : 5-gcugauggcgaugaaugaacacugcguuugcuggcuuugaugaaa-3. 5 rlm - race outer primer : 5-gctgatggcgatgaatgaacactg-3. 5 rlm- race inner primer : 5-cgcggatccgaacactgcgtttgctggctttgatg-3. srna - sequencing for 21-nt and 24-nt srnas at transposons in col-0 , ddm1 - 2 , rdr6 - 15 , ddm1 - 2 rdr6 - 15 ( hiseq ) and col-0 , ddm1 - 2 and ddm1 - 2 dcl1 - 11 ( miseq ) . normalized reads per million ( rpm ) for srna reads , unique ( u ) mapping to one location of the genome , and multiple ( m ) mapping to more than one location of the genome . exact binomial two - sided test given for transposon methylation in ddm1 - 2 rdr6 - 15 replicates as compared to ddm1 - 2 . p - value < 2.2e-16 alternative hypothesis : true probability of success is not equal to 0.5 , 95 % confidence interval , sample estimates : probability of success given . known and predicted mirna abundance , sequence , length ( nt ) and rna type indicating if new mirna length at known immature mirna precursor for col-0 , ddm1 - 2 , rdr6 - 15 and ddm1 - 2 rdr6 - 15 srna - seq libraries . analysis of pare libraries by utilizing the predicted mirna binding site within each transposon - coded open - reading frame ( orf ) . mirna sequence and length are shown for each target orf ( atx ) along with the co - ordinate and genomic dna strand ( + / ) of the predicted cleavage site . 31-nt ( l ) windows at the mirna binding site are shown in col-0 , ddm1 - 2 , rdr6 - 15 and ddm1 - 2 rdr6 - 15 ( refer to supplementary methods for details ) , followed by the genomic co - ordinates and strand ( + / ) of the target orf . cleavage products were detected from the same strand or different strand as the target orf , depending whether cleavage was of the sense or antisense transcript , respectively . for statistical significance of the pare reads at the mirna cleavage site , we performed the chi - square goodness of fit test , calculating the p - value for observed value given the expected value . if we assume pare tags fall randomly on all the positions in the long window ( 31-nt ) , the expected frequency for pare reads fall into the small window ( 5-nt ) would be 5/31 , therefore , the expected frequency would be would 26/31 for other regions . dna methylation by cytosine context ( cg , chg and chh ) calculated per transposon by overall count of cytosine to thymine at each cytosine position ( 1 = methylated cytosine , 0 = unmethylated cytosine ) for each bisulphite sequencing library : col-0 , ddm1 - 2 , rdr6 - 15 and two replicates of ddm1 - 2 rdr6 - 15 . expression of tes by fold change normalized to col-0 wt , in ddm1 - 2 , rdr6 - 15 and ddm1 - 2 rdr6 - 15 by log2 affymetrix microarray signal intensity . for known mirnas targeting tes passing stringent criteria based on pare , confirming targeting , both abundance and strand orientation is given at transposons . ( refer to methods summary , to accompany table 1 , adapted from supplementary table 3 ) . epigenetically - activated mirnas with relaxed criteria for pare cleavage data , confirming targeting , with both abundance and strand orientation given at tes . ( refer to methods summary , to accompany table 1 , adapted from supplementary table 3 ) . read count for srna - seq , bis - seq and pare libraries used in this study , mapped to the col-0 reference genome ; tair10 ( refer to methods summary for computational details ) . analysis of pare libraries by utilizing the predicted mirna binding site within each transposon - coded open - reading frame ( orf ) . mirna sequence and length are shown for each target orf along with the co - ordinate and genomic dna strand ( + / ) of the predicted cleavage site . pare normalized read abundance ending within the shorter small 1-nt ( ss ) and shorter long 3-nt ( sl ) windows at the mirna binding site are shown in col-0 , ddm1 - 2 , rdr6 - 15 and ddm1 - 2 rdr6 - 15 ( refer to supplementary methods for details ) , followed by the genomic coordinates and strand ( + / ) of the target orf . cleavage products were detected from the same strand or different strand as the target orf , depending whether cleavage was of the sense or antisense transcript , respectively .
in plants , post - transcriptional gene silencing ( ptgs ) is mediated by dicer - like1 ( dcl1)-dependent mirnas , that also trigger 21-nt secondary sirna via rna dependent rna polymerase6 ( rdr6 ) , dcl4 , and argonaute1 ( ago1)13 , while transcriptional gene silencing ( tgs ) of transposons is mediated by 24-nt heterochromatic ( het)sirna rdr2 , dcl3 and ago44 . 
 transposons can also give rise to abundant 21-nt  epigenetically activated  small interfering rnas ( easirnas ) in decrease in dna methylation1 ( ddm1 ) and dna methyltransferase1 ( met1 ) mutants , as well as in the vegetative nucleus of pollen grains5 , and in dedifferentiated plant cell cultures6 . 
 here we show that easirnas resemble secondary sirnas , in that thousands of transposon transcripts are specifically targeted by more than fifty mirnas for cleavage and processing by rdr6 . 
 loss of rdr6 , dcl4 or dcl1 in a ddm1 background results in loss of 21-nt easirna , and severe infertility , but 24-nt hetsirna are partially restored , supporting an antagonistic relationship between ptgs and tgs . 
 thus mirna - directed easirna biogenesis is a latent mechanism that specifically targets transposon transcripts , but only when they are epigenetically reactivated during reprogramming of the germline . 
 this ancient recognition mechanism may have been retained both by transposons to evade long - term heterochromatic silencing , and by their hosts for genome defence .