The MET1b gene encoding a maintenance DNA methyltransferase is indispensable for normal development in rice
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While Arabidopsis bears only one MET1 gene encoding the DNA methyltransferase that is mainly responsible for maintaining CG methylation after DNA replication, rice carries two MET1 genes, MET1a and MET1b, expressed in actively replicating and dividing cells, and MET1b is more abundantly expressed than is MET1a. A met1a null mutant displayed no overt phenotypes, implying that MET1b must play a major role in the maintenance DNA methylation. Here, we employed two met1b null mutants, generated by homologous recombination-mediated knock-in targeting and insertion of endogenous retrotransposon Tos17. These MET1a/MET1a met1b/met1b homozygotes exhibited abnormal seed phenotypes, which is associated with either viviparous germination or early embryonic lethality. They also displayed decreased levels of DNA methylation at repetitive CentO sequences and at the FIE1 gene locus in the embryos. In addition, independently isolated knock-in-targeted plants, in which the promoterless GUS reporter gene was fused with the endogenous MET1b promoter, showed the reproducible, dosage-dependent, and spatiotemporal expression patterns of GUS. The genotyping analysis of selfed progeny of heterozygous met1a met1b null mutants indicated that weakly active MET1a seems to serve as a genetic backup mechanism in rice met1b gametophytes, although the stochastic and uncoordinated activation of epigenetic backup mechanisms occurred less efficiently in the met1b homozygotes of rice than in the met1 homozygotes of Arabidopsis. Moreover, passive depletion of CG methylation during the postmeiotic DNA replication in the haploid nuclei of the met1a met1b gametophytes in rice results in early embryonic lethality. This situation somewhat resembles that of the met1 gametophytes in Arabidopsis.
KeywordsKnock-in Gene targeting Tos17 insertion mutants GUS reporter gene METHYLTRANSFERASE 1 (MET1) Rice (Oryza sativa)
We thank Hisayo Asao, Miki Shimamoto, and Miwako Matsumoto for their technical assistance; Akemi Ono, Satoru Moritoh, Atsushi Hoshino, Kazuo Tsugane, and Yutaka Sato for discussion; Masahiro Mii, Mikio Nakazono, Hirokazu Kobayashi, and Hiroshi Noguchi for their encouragement; and the Rice Genome Resource Center for providing Tos17 insertion mutant lines. This work was supported by grants from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) from Bio-oriented Technology Research Advancement Institution (BRAIN) in Japan (to SI) and from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) (No. 2237001 to SI, No. 18-40089 to YJ-H). SI and YJ-H were supported by a grant from the Global Center of Excellence program of MEXT, and YJ-H was also supported by an RPD fellowship from the Japan Society for the Promotion of Science (JSPS).
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