IDN2 has a role downstream of siRNA formation in RNA-directed DNA methylation.
In plants, a particular class of short interfering (si)RNAs can serve as a signal to induce cytosine methylation at homologous genomic regions. If the targeted DNA has promoter function, this RNA-directed DNA methylation (RdDM) can result in transcriptional gene silencing (TGS). RNA-directed transcriptional gene silencing (RdTGS) of transgenes provides a versatile system for the study of epigenetic gene regulation. We used transcription of a nopaline synthase promoter (ProNOS)-inverted repeat (IR) to provide a RNA signal that triggers de novo cytosine methylation and TGS of a homologous ProNOS copy in trans. Utilizing a ProNOS-NPTII reporter gene showing high sensitivity to silencing in this two component system, a forward genetic screen for EMS-induced no rna-directed transcriptional silencing (nrd) mutations was performed in Arabidopsis thaliana. Three nrd mutant lines were found to contain one novel loss-of-function allele of idn2/rdm12 and two of nrpd2a/nrpe2a. IDN2/RDM12 encodes a XH/XS domain protein that is able to bind double-stranded RNA with 5' overhangs, while NRPD2a/NRPE2a encodes the common second-largest subunit of the plant specific DNA-dependent RNA polymerases IV and V involved in silencing processes. Both idn2/rdm12 and nrpd2a/nrpe2a release target transgene expression and reduce CHH context methylation at transgenic as well as endogenous RdDM target regions to similar extents. Nevertheless, accumulation of IR-derived siRNA is not affected, allowing us to present a refined model for the pathway of RdDM and RdTGS that positions function of IDN2 downstream of siRNA formation and points to an important role for its XH domain.
Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany.
Andreas
A
Finke
Markus
M
Kuhlmann
Michael Florian
MF
Mette
D000594:Amino Acid Oxidoreductases / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D017930:Genes, Reporter; D055029:Inverted Repeat Sequences; D009154:Mutation; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D029681:Arabidopsis Proteins; C545931:IDN2 protein, Arabidopsis; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase; D012321:DNA-Directed RNA Polymerases