Paternal easiRNAs establish the triploid block in Arabidopsis

Polyploidy, the presence of more than two sets of chromosomes within the nucleus, is a common phenomenon among plants that has shaped genome organization and is thought to be a major driver of speciation. The triploid block acts as a reproductive barrier that prevents successful backcrosses of newly formed polyploids with their progenitors. It is established in the endosperm, an ephemeral tissue that nurtures the developing embryo. Here we show that paternal 21/22 nucleotide epigenetically activated small interfering RNAs (easiRNAs) in Arabidopis thaliana are responsible for the establishment of the triploid block associated seed abortion. This dramatic phenotype is overcome when crosses are performed with a paternal mutant in the plant specific RNA polymerase IV (Pol IV). Loss of Pol IV reduces both easiRNAs and hetsiRNAs in the pollen grain. Seeds derived from crosses between wild type tetraploid fathers and diploid mothers have reduced levels of hetsiRNAs and cytosine methylation at transposable elements (TEs), indicating that they have suffered an excess of epigenetic reprogramming due to an excess of paternally derived easiRNAs. Paternal Pol IV mutants are able to buffer that easiRNA excess and rescue the abortion phenotype by rescuing both the cytosine methylation level at TEs and the 24 nt hetsiRNA accumulation levels. This mechanism highlights the elegant and important TE regulation mechanism controlled by the plant specific Pol IV that takes place pre-fertilization and regulates paternal easiRNAs and maternal hetsiRNAs, and determines the post-fertilization epigenetic stability and seed viability. 15 small RNA sequencing libraries, 8 genome-wide cytosine methylation libraries and 8 RNA-seq libraries. 7 small RNA libraries were generated from mature pollen grains of diploid and tetraploid Col-0 wt and nrpd1a mutants (1n and 2n pollen, respectively) and Col-0 wt and the mutant backgrounds dcl4 rdr6 and rdr6. 8 small RNA libraries were generated from seeds at 6 days after pollination from the cross of maternal Ler with paternal Col-0 wt and the mutant backgrounds osd1,nrpd1a, and nrpd1a osd1. 8 cytosine methylation libraries were generated from hand-dissected endosperm at 6-7 days after pollination from the cross of maternal Ler with paternal Col-0 wt and the mutant backgrounds osd1,nrpd1a, and nrpd1a osd1. 8 RNA-seq libraries were generated from hand-dissected endosperm at 6-7 days after pollination from the cross of maternal Ler with paternal Col-0 wt and the mutant backgrounds osd1,nrpd1a, and nrpd1a osd1.