Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.NIH grant: (R01 GM067014); Temasek Lifescience Laboratory; NSERC graduate student fellowship; Fred C. Gloeckner Foundation; Belgian American Educational Foundation postdoctoral fellowship: (Herbert Hoover)

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)1–3, 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.

Chromosome dosage plays a significant role in reproductive isolation and speciation in both plants and animals, but underlying mechanisms are largely obscure1. Transposable elements can promote hybridity through maternal small RNA2, and have been postulated to regulate dosage response via neighboring imprinted genes3,4. Here, we show that a highly conserved microRNA in plants, miR845, targets the tRNAMet primer-binding site (PBS) of LTR-retrotransposons in Arabidopsis pollen, and triggers the accumulation of 21 to 22-nucleotide small RNA in a dose dependent fashion via RNA polymerase IV. We show that these epigenetically activated small-interfering RNAs (easiRNAs) mediate hybridization barriers between diploid seed parents and tetraploid pollen parents (“the triploid block”), and that natural variation for miR845 may account for “endosperm balance” allowing formation of triploid seeds. Targeting the PBS with small RNA is a common mechanism for transposon control in mammals and plants, and provides a uniquely sensitive means to monitor chromosome dosage and imprinting in the developing seed.