Mutation of the RDR1 gene caused genome-wide changes in gene expression, regional variation in small RNA clusters and localized alteration in DNA methylation in rice.

Ningning Wang,Bao Liu,Xiuyun Lin,Hui Wang,Xiufang Ou,Hongwei Xun,Ying Liu,Zhenhui Wang,Wei Huang,Chunyu Zhang,Ming-Bo Wang,Ning Li,Jian Ma,Di Zhang

doi:10.1186/1471-2229-14-177

Abstract

BackgroundEndogenous small (sm) RNAs (primarily si- and miRNAs) are important trans/cis-acting regulators involved in diverse cellular functions. In plants, the RNA-dependent RNA polymerases (RDRs) are essential for smRNA biogenesis. It has been established that RDR2 is involved in the 24 nt siRNA-dependent RNA-directed DNA methylation (RdDM) pathway. Recent studies have suggested that RDR1 is involved in a second RdDM pathway that relies mostly on 21 nt smRNAs and functions to silence a subset of genomic loci that are usually refractory to the normal RdDM pathway in Arabidopsis. Whether and to what extent the homologs of RDR1 may have similar functions in other plants remained unknown.ResultsWe characterized a loss-of-function mutant (Osrdr1) of the OsRDR1 gene in rice (Oryza sativa L.) derived from a retrotransposon Tos17 insertion. Microarray analysis identified 1,175 differentially expressed genes (5.2% of all expressed genes in the shoot-tip tissue of rice) between Osrdr1 and WT, of which 896 and 279 genes were up- and down-regulated, respectively, in Osrdr1. smRNA sequencing revealed regional alterations in smRNA clusters across the rice genome. Some of the regions with altered smRNA clusters were associated with changes in DNA methylation. In addition, altered expression of several miRNAs was detected in Osrdr1, and at least some of which were associated with altered expression of predicted miRNA target genes. Despite these changes, no phenotypic difference was identified in Osrdr1 relative to WT under normal condition; however, ephemeral phenotypic fluctuations occurred under some abiotic stress conditions.ConclusionsOur results showed that OsRDR1 plays a role in regulating a substantial number of endogenous genes with diverse functions in rice through smRNA-mediated pathways involving DNA methylation, and which participates in abiotic stress response.

Highlights

IntroductionEndogenous small (sm) RNAs (primarily si- and micro RNAs (miRNAs)) are important trans/cis-acting regulators involved in diverse cellular functions
Endogenous small RNAs are important trans/cis-acting regulators involved in diverse cellular functions
Semi-quantitative and real-time quantitative (q)reverse transcriptase (RT)-PCR analyses confirmed that the homozygous OsRDR1 mutant (Osrdr1) had a complete loss of OsRDR1 expression in shoot-tip tissue wherein the gene was highly expressed in wild type (WT) plants (Figure 1c)

Summary

Introduction

Endogenous small (sm) RNAs (primarily si- and miRNAs) are important trans/cis-acting regulators involved in diverse cellular functions. 21–22 nt siRNAs guide the degradation of viral RNAs as well as some endogenous mRNAs and are important for plant defense against viruses and for some aspects of plant development [4,5,6] Unlike these PTGSassociated smRNAs, the 24 nt siRNAs are associated with RNA-directed DNA methylation (RdDM), a plantspecific de novo DNA methylation pathway required for transcriptional silencing of transposable elements and other DNA repeats to maintain genome stability [7,8,9,10]. Rdr mutant of Arabidopsis showed loss of DNA methylation in a subset of genomic loci in comparison to wild-type Arabidopsis plants [20,21], suggesting that RDR1 plays a role in the recently identified non-canonical, 21 nt siRNA-directed RdDM pathway [20,21]. The function of RDR1 in gene regulation from a genome-wide perspective has not been investigated in any plant

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