Characterization of two rice DNA methyltransferases and RNAi-mediated restoration of promoter activity in silenced rice callus

Two genomic clones (OsMET1-1, AF 462029 and OsMET1-2), each encoding a cytosine-5 DNA methyltransferase (MTase), were isolated from rice (Oryza sativa L.) BAC libraries. OsMET1-1 has an open reading frame of 4,566 nucleotides with twelve exons and eleven introns while OsMET1-2 has an open reading frame of 4,452 nucleotides with eleven exons and ten introns. Although OsMET1-1 and OsMET1-2 have high sequence similarity overall, they share only 24% identity in exon 1 and intron 3 of OsMET1-1 is absent from OsMET1-2. As for other eukaryotic DNA MTases of the Dnmt1/MET l class, the derived amino acid sequences of OsMET1-1 and OsMET1-2 suggest that they are comprised of two-thirds regulatory domain and one-third of catalytic domain. Most functional domains identified for other MTases were present in the rice MET1 sequences. Amino acid sequence comparison indicated high similarity (56-75% identity) of rice MET1s to other plant MET1 sequences but limited similarity (~24% identity) to animal Dnmt1s. Genomic blot and database analysis indicated the presence of a single copy of OsMET1-1 (on chromosome 3) and single copy of OsMET1-2 (on chromosome 7). Ribonuclease protection assays revealed expression of both OsMET1-1 and OsMET1-2 in highly dividing cells, but the steady state level of OsMET1-2 was 7-12 fold higher than that for OsMET1-1 in callus, root and inflorescence. The functional involvement of the rice DNA MTases in gene silencing was investigated using an RNAi strategy. Inverted repeat constructs of either the Nor C-terminal regions of OsMET1-1 were supertransformed into calli derived from a rice line bearing a silenced 35S-uidA-nos transgene. Restoration of uidA expression in the bombarded calli was consistent with the inactivation of maintenance methylation and with previous evidence for the involvement of methylation in silencing of this line.