Abstract
To properly regulate the genome, cytosine methylation is established by animal DNA methyltransferase 3 s (DNMT3s). While altered DNMT3 homologs, Domains rearranged methyltransferases (DRMs), have been shown to establish methylation via the RNA directed DNA methylation (RdDM) pathway, the role of true-plant DNMT3 orthologs remains elusive. Here, we profile de novo (RPS transgene) and genomic methylation in the basal plant, Physcomitrella patens, mutated in each of its PpDNMTs. We show that PpDNMT3b mediates CG and CHH de novo methylation, independently of PpDRMs. Complementary de novo CHG methylation is specifically mediated by the CHROMOMETHYLASE, PpCMT. Intragenomically, PpDNMT3b functions preferentially within heterochromatin and is affected by PpCMT. In comparison, PpDRMs target active-euchromatic transposons. Overall, our data resolve how DNA methylation in plants can be established in heterochromatin independently of RdDM; suggest that DRMs have emerged to target euchromatin; and link DNMT3 loss in angiosperms to the initiation of heterochromatic CHH methylation by CMT2.