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

Hybridization of plants that differ in number of chromosome sets (ploidy) frequently causes endosperm failure and seed arrest, a phenomenon referred to as triploid block. Mutation in NRPD1 , encoding the largest subunit of the plant-specific RNA Polymerase IV (Pol IV), can suppress the triploid block. Pol IV generates short RNAs required to guide de novo methylation in the RNA-directed DNA methylation (RdDM) pathway. In this study, 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. Inbreeding of nrpd1 was connected with a transgenerational loss of non-CG DNA methylation on sites jointly regulated by CHROMOMETHYLASES 2 and 3. 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. One-sentence summary Inbreeding of mutants impaired in RdDM components transgenerationally enhanced their ability to suppress the triploid block.