Transgenerational effect of mutants in the RNA-directed DNA methylation pathway on the triploid block

Hybridizations of plants that differ in number of chromosome sets (ploidy) frequently causes endosperm failure and seed arrest, a phenomenon referred to as triploid block. Unreduced diploid gametes generated by the omission of second division1 (osd1) mutant induce the triploid block, similar as tetraploid (4x) plants. We recently found that mutations in NRPD1, encoding the largest subunit of the plant-specific RNA Polymerase IV (Pol IV), can suppress the triploid block. Pol IV generates small RNAs required to guide de novo methylation in the RNA-directed DNA methylation (RdDM) pathway. Strikingly however, mutations in other components of the RdDM pathway like RDR2 and NRPE1 fail to suppress the triploid block when inherited in the osd1 background, but have a suppressive effect as 4x mutants. In this study, we aimed at understanding the cause for this discrepancy. We found that the ability of mutants in the RdDM pathway to suppress the triploid block depends on their degree of inbreeding. While nrpd1 is able to suppress in the first homozygous generation, mutants in RDR2, NRPE1, and DRM2 require at least one additional round of inbreeding to exert a suppressive effect. Our data thus reveal that loss of RdDM function differs in its effect in early and late generations and that Pol IV acts at an early stage of triploid block establishment. 6 genome-wide cytosine methylation libraries and 6 ChIP sequencing libraries. 6 genome-wide cytosine methylation libraries were generated from Arabidopsis leaves of wt, F2 and Fi nrpd1 mutants. 6 ChIP sequencing libraries were generated from Arabidopsis leaves of wt and nrpd1 mutants.