BACKGROUND
rna silencing is an evolutionally conserved gene regulation mechanism in eukaryotes mediated by 20– <dig> nt non-coding small rnas.
these smrnas are processed from double-stranded  or hairpin rna molecules by dicer-like  proteins, and guide rna-induced silencing complexes to cognate single-stranded rnas based on sequence complementarity, and result in degradation of the targeted rnas  <cit> .
in plants, there are several different classes of smrnas, including 20– <dig> nt micro rnas  processed by dcl <dig>  21– <dig> nt small interfering rnas  by dcl <dig> and dcl <dig>  and the  <dig> nt heterochromatin-associated sirnas by dcl <dig>  mirnas play an important role in plant development by directing posttranscriptional gene silencing  of regulatory genes such as those encoding transcription factors.
similarly, 21– <dig> 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  <cit> .
unlike these ptgs-associated smrnas, the  <dig> nt sirnas are associated with rna-directed dna methylation , a plant-specific de novo dna methylation pathway required for transcriptional silencing of transposable elements and other dna repeats to maintain genome stability  <cit> .
the biogenesis of sirnas in plants requires the activity of rna-dependent rna polymerase , which converts single-stranded rnas to dsrna precursors of sirnas.
the dicot model plant arabidopsis thaliana has six rdr genes, i.e., rdr <dig>  rdr <dig>  rdr3a, rdr3b, rdr3c and rdr <dig> <cit> , of which three rdrs  are shown to play roles in the rna silencing pathways.
rdr <dig> is required for 24 nt sirna biogenesis and therefore involved in the canonical rddm pathway  <cit> .
rdr <dig> is involved in the production of the endogenous 21 nt trans-acting sirnas and also essential for sense transgene-induced ptgs  <cit> .
both rdr <dig> and rdr <dig> are also involved in viral sirna accumulation in infected arabidopsis plants <cit> .
the function of rdr <dig> in rna silencing is less understood, but recent studies have shown that it is involved in sirna biogenesis from a subset of rna viruses  <cit> .
furthermore, rdr <dig> mutant of arabidopsis showed loss of dna methylation in a subset of genomic loci in comparison to wild-type arabidopsis plants  <cit> , suggesting that rdr <dig> plays a role in the recently identified non-canonical, 21 nt sirna-directed rddm pathway  <cit> .
however, the function of rdr <dig> in gene regulation from a genome-wide perspective has not been investigated in any plant.
in contrast to arabidopsis that has six rdr genes, the rdr family of rice , a model plant for monocots, contains only three members, namely osrdr <dig>  osrdr <dig> and osrdr <dig> <cit> .
a previous study showed that osrdr <dig> has a similar function to its counterparts in arabidopsis and tobacco  in ptgs-based silencing of certain rna viruses, such as bromovirus  <cit> .
to investigate if osrdr <dig> plays a role in regulation of endogenous genes in rice, we characterized a loss-of-function mutant of osrdr <dig> derived from a disruptive ltr retrotransposon  insertion into the 2nd exon of the gene.
we investigated genome-wide changes in gene expression and smrna profiles, localized changes in dna methylation, and phenotypes under normal and several abiotic stress conditions in this rice rdr <dig> mutant.
RESULTS
characterization of the rice rdr <dig> mutant
we obtained a ltr retrotransposon tos <dig> <cit>  insertion line for osrdr <dig>  from the tos <dig> insertion mutant library of rice cv.
hitomebore .
molecular characterization identified h <dig> as heterozygous for a tos <dig> insertion into the second exon of osrdr <dig> .
we obtained the homozygous mutant  and its sibling wild type  plants by selfing of the heterozygous plant  for five successive generations.
in each generation, the three kinds of genotypes, wt, heterozygote and homozygous mutant, were selected based on locus-specific pcr amplifications .
both the heterozygous and homozygous plants for osrdr <dig> showed no discernibly altered phenotypes in the entire growth and developmental period over multiple generations under normal field conditions .
semi-quantitative and real-time quantitative rt-pcr analyses confirmed that the homozygous osrdr <dig> mutant  had a complete loss of osrdr <dig> expression in shoot-tip tissue wherein the gene was highly expressed in wt plants .
this indicated that the exonic insertion of tos <dig> knocked out the expression of osrdr <dig>  and hence, abolished its function.
genome-wide changes in gene expression in osrdr1
we profiled the transcriptome of shoot-tip tissues between osrdr <dig> and its sibling wt plants using the affymetrix genechip rice® genome array .
after normalization of the microarray data, we detected  <dig>  expressed genes in the shoot-tip tissue of rice.
the expression levels of  <dig>  genes were conserved between osrdr <dig> and wt, but  <dig> and  <dig> genes showed significant up- and down-regulation in osrdr <dig>  respectively .
a gene ontology  category analysis of these  <dig>  differentially expressed genes showed that they were enriched in a variety of go categories .
however, these  <dig>  differentially expressed genes were found to distribute non-randomly across the  <dig> rice chromosomes .
for example, chromosomes  <dig>   <dig> and  <dig> contained significantly more distributions than the rest chromosomes .
it is also clear from the data that within a given chromosome, the distribution is also nonrandom, for example, the distributions are almost exclusively confined to the long arms of chromosomes  <dig> and  <dig> and  <dig> relative to their respective short-arms .
the highly reproducible microarray profiles among three biological replicates for both osrdr <dig> and its wt sibling plants testified reliability of the data and their analysis.
all microarray data have been submitted to the geo repository under the accession number of gse <dig>  to further verify the quality of the microarray data and analysis, we analyzed  <dig> genes representing both up- and down-regulation in osrdr <dig> vs. wt, as well as equal expression between the two lines using qrt-pcr assay on the same cdnas as used for microarray.
the qrt-pcr results were highly consistent with the microarray data for almost all the  <dig> tested genes in levels or at least in trends of expression changes , confirming reliability of the microarray analysis.
alteration in smrna clusters in osrdr1
previous studies have established that rdr <dig> function is required for biogenesis and/or amplification of some types of rna virus-related smrna accumulation in arabidopsis <cit>  and tobacco   <cit> .
these findings promoted us to test whether loss of function of osrdr <dig> may have a general impact on “normal” smrna abundance in rice, and we investigated this issue by high-throughput smrna sequencing.
comparison of the  <dig> , <dig> clean smrna reads from osrdr <dig> with the  <dig> , <dig> reads from its sibling wt  revealed highly similar profiles in both size distributions and sequence categories of the smrnas between osrdr <dig> and wt , suggesting that the overall smrna abundance was not generally affected by the loss of function of osrdr <dig>
genome-wide overall similarity in smrna abundance does not necessitates absence of smrna fluctuations in localized smrna clusters, because up- and down-regulated smrna accumulation can be masked by reciprocal compensation.
we thus investigated localized smrna accumulation between osrdr <dig> and its sibling wt by mapping the cleaned smrna reads to 100 bp sliding windows  across each of the  <dig> rice chromosomes, normalized the reads to reads per million , and then compared the rpm smrna clusters between osrdr <dig> and wt.
using 4-fold difference as a cut-off threshold, we identified many smrna clusters with differential abundance between osrdr <dig> and its sibling wt, which were uniformly distributed across the entire length of each chromosome .
next, we extracted the differentially expressed smrnas between osrdr <dig> and wt  in the size range of 20-24 nt, which should parsimoniously contain all sirnas, and mapped them to the same 100 bp windows across each chromosome.
we found that this subset of differential smrna clusters also distribute on both arms of each chromosome , although due to their smaller numbers, we cannot rule out the possibility that the distribution might show “hot spots” within a given chromosome.
taken together, the smrna sequencing data suggested that loss-of-function mutation in osrdr <dig> caused extensive alterations in smrna clusters across each chromosome and throughout the genome, but it did not result in marked fluctuations of overall smrna profiles, probably due to more or less equally increased and decreased abundance of the smrna clusters which offset each other.
altered expression of mirnas and their target genes in osrdr1
given the diverse important roles played by mirnas, we investigated if their accumulation might be affected in the osrdr <dig> mutant.
previous computational and cloning studies have identified ca.
<dig> mirnas from  <dig> mirna families in rice  <cit> .
based on this information, we first analyzed the abundance of known rice mirnas   in osrdr <dig> and wt.
this analysis  indicated that:  majority  of the known osa-mirnas were expressed equally or nearly so between osrdr <dig> and wt;  some mirnas  showed >  <dig> fold increased expression in osrdr <dig> relative to wt, with the highest expression ratio reaching  <dig> : <dig> ;  some mirnas  showed > <dig> fold decrease in expression in osrdr <dig> relative to wt, with the lowest expression ratio of 1: <dig>  observed for mir167j between mutant and wt ;  osa-mir395p and osa-mir395s, being from the same mirna family, showed changes in expression to opposite directions, with an expression ratio of 1: <dig>  for osa-mir395p but  <dig> : <dig> for osa-mir395s in mutant vs. wt .
to verify the expression differences based on the smrna sequencing data, we performed semi-nested qrt-pcr analysis of four mirnas in mutant and wt.
the qrt-pcr results were found consistent with the smrna sequencing data , confirming the changes in mirna expression between osrdr <dig> and wt.
in addition to the known mirnas, we identified a total of  <dig> putative novel osa-mirnas from the smrna data of the mutant and wt plants based on prediction of pre-mirna-like stem-loop structures in sequences surrounding the smrna sequences in the rice genome .
three of these novel mirnas  had an identical mature sequence but corresponded to three independent genomic loci, thereby forming a novel mirna family.
for the putative mirna osr-mirna-n <dig>  smrna reads were detected from both the 5’  and the 3’  half of the predicted stem-loop structure, but the 5p smrna showed a higher abundance than the 3p smrna , indicating that the 5p smrna is the guide strand  whereas the 3p smrna is the passenger strand   <cit> .
like the known mirnas, these novel mirnas also showed expression variation between osrdr <dig> and wt, with  <dig> showing expression only in osrdr <dig>  and  <dig> showing expression only in wt plants .
taken together, our results suggest that osrdr <dig> was likely involved in mirna accumulation in rice.
to investigate if the altered mirna accumulation in osrdr <dig> relative to wt was associated with changes in mirna target gene expression, we compared the mirna expression profiles  derived from the smrna sequencing data with the target gene expression levels based on the microarray data.
we did not find a generalized relationship between the mirna abundance and target gene expression levels .
instead, four types of relationships were recognized for a subset of mirnas and their predicted targets , which included:  reduced mirna abundance was correlated with up-regulated expression of target genes in osrdr <dig> relative to wt ;  increased mirna abundance was correlated with down-regulated expression of target genes in osrdr <dig> relative to wt ;  both mirnas and their target genes were up-regulated in osrdr <dig> relative to wt ;  both mirnas and their target genes were down-regulated in osrdr <dig> relative to wt .
the first two types of relationships supported a role of mirnas in down-regulating expression of their predicted target genes.
the last two types of relationships could be a result of concordant transcriptional regulation of the mirnas and their target genes caused by another more upstream regulator whose expression or activity was modified due to loss of function of osrdr <dig>  all small rna data have been submitted to genbank under the accession numbers of srp <dig>
locus-specific alteration of dna methylation in osrdr1
as the arabidopsis rdr <dig> has been shown to play a role in the non-canonical, nerd-dependent rddm pathway  <cit> , we were interested to know if osrdr <dig> might have a similar function in rice.
we therefore examined cytosine methylation and gene expression levels of  <dig> selected genomic loci in osrdr <dig> and its sibling wt using bisulphite sequencing and qrt-pcr analysis.
these loci overlapped with two transposable elements  and three protein-coding genes, which were chosen as representatives because they all showed alteration in smrna clusters in osrdr <dig> relative to wt .
the bisulphite-sequenced regions for the two tes  included:  portions of the 5’- and 3’-ltrs together with their immediate flanking regions of two tos <dig> copies  ;  the 5’ termini along with their immediate flanking regions of two pong copies  , and;  a body-region of the transposase-encoding orf of pong  that is shared by all conserved copies of the element.
the bisulphite-sequenced regions of the three protein-coding genes are all within their 5’-upstream regions .
the bisulphite sequencing results showed that:  of the five tos <dig> regions analyzed, only the 5’ ltr region for the tos <dig> copy located on chromosome  <dig> showed marked decrease  in cg and chg methylation but not in chh methylation in osrdr <dig> relative to wt ;  for the three pong regions analyzed, only the 5’region of the copy located on chromosome  <dig> showed clear methylation changes: decrease in cg methylation by 20% and increase in both chg and chh methylation by approximately 30% and 50%, respectively, in osrdr <dig> relative to wt ; of the three genic loci analyzed, only one  showed increase in cg methylation by ca.
50% in osrdr <dig> relative to wt, while methylation of the other two regions were unchanged .
the two tes showed significant up-regulation in osrdr <dig> relative to wt , consistent with a decrease in methylation at the 5’ regions of one copy of each tes in osrdr <dig> .
notably, all the three genes analyzed did not show the expected relationship between dna methylation state of their 5’-regulatory regions and expression levels.
specifically, one gene  that showed increase in cg methylation in osrdr <dig> was up-regulated in expression ; the remaining two genes showed down-regulation in osrdr <dig> relative to wt despite the lack of methylation changes in the bisulphite-sequenced regions .
we next investigated possible relationships between smrna accumulation and dna methylation.
we found that almost all of the altered chh methylation was associated with changes in smrna clusters.
for example, the increased chh methylation of the pong copy located on chromosome  <dig> was associated with a moderate increase in smrna accumulation, whereas the slight decrease in chh methylation in the flanking region and the increase in chh methylation in the gene body region of the pong copy located on chromosome  <dig> were associated with moderate decrease and increase in smrna accumulations, respectively .
these positive correlations of smrna accumulation and chh methylation suggests that osrdr <dig> plays a role in the de novo chh methylation in a subset of genomic loci in rice, probably by affecting the production/accumulation of smrnas required for rddm, as shown in arabidopsis <cit> .
the locus-specificity of methylation changes or the two analyzed tes indicated that their methylation patterns were determined by either or both the flanking sequences and the local chromatin environment, an issue which warrants further investigations.
phenotypes in osrdr <dig> under normal and abiotic stress conditions
it is known that various stress conditions may produce protracted effects on genome stability, leading to transgenerational changes in genome structure, which are proposed to have been initiated by epigenetic mechanisms  <cit> .
we were therefore interested to know if osrdr <dig> may play a role in stress response in rice.
we quantified phenotypes between osrdr <dig> and wt plants under normal and several short-term abiotic stress conditions , which included treatments with salt, heavy metals cu2+and hg2+, and overdose nitric oxide .
the results showed that no phenotypic difference was found between osrdr <dig> and wt under normal condition, but significant ephemeral phenotypic differences between the two genotypes emerged in some of the different stress conditions .
specifically,  seedlings of osrdr <dig> were more sensitive than wt to salt and overdose no treatments, as being reflected by reduced plant height, root length and biomass at the seedling stage, with the difference in plant height and root length being persisted to the heading stage after removal of the stresses;  seedlings of osrdr <dig> showed increased tolerance to heavy metal cu2+/hg2+ as indicated by increased root length, but upon removal of the stresses the differences were gradually attenuated and completely disappeared at the heading stage;  when both unstressed and the transiently-stressed plants of the mutant and wt were grown to maturity, no difference in plant height, tiller number, panicle and kernel traits was observed between the two genotypes.
collectively, our results suggest that osrdr <dig> has a potential function in stress response in rice, but the effects are contingent with presence of stresses without exerting protracted influence when the stresses are removed.
discussion
rna silencing pathways have been well characterized in the dicot model plant arabidopsis but remain poorly studied in other plants like monocots to which many major crops belong.
utilizing a retransposon tos <dig> insertion mutant of osrdr <dig> in rice, we performed genome-wide analysis to unveil the function of osrdr <dig>  a component recently shown in arabidopsis to be involved in non-canonical, 21 nt sirna-directed rddm pathway  <cit> , on expression of endogenous genes.
by deep sequencing of smrnas and microarray analysis of gene expression in the osrdr <dig> mutant and its sibling wt, we showed that the expression of >  <dig>  genes were significantly changed in osrdr <dig> relative to wt, suggesting that osrdr <dig> plays a role in genome-wide gene regulation in rice.
in addition, the osrdr <dig> mutant showed regional alterations in smrna accumulation and/or titration across the rice genome, and at least some of which are associated with locus-specific alteration of dna methylation.
among the differentially accumulated smrnas, many were mirnas both previously known and newly identified in this study.
expression changes in many of these mirnas are associated with changes in their target gene expression, which could partly be responsible for the gene expression changes observed in the osrdr <dig> mutant relative to wt.
the mechanism by which mutation of osrdr <dig> caused changes in mirna expression in rice was not clear.
it was found in arabidopsis that none of the rdrs has a direct role in mirna biogenesis  <cit> .
however, rdrs could impact mirna accumulation indirectly by either affecting mirna precursor gene expression through the tgs or ptgs pathways  <cit>  or generating dsrnas that compete for dcl <dig> function that is required for mirna biogenesis  <cit> .
our results suggested that osrdr <dig> might play a role in maintaining the intrinsic locus-specific dna methylation patterns, as its mutation caused alteration of methylation at some of the loci we analyzed.
in particular, the changes in chh methylation, which are indicative of de novo methylation by the rddm pathway, showed correlation with changes in smrna accumulation.
in the respect of reduced chh methylation and concomitant reduced smrna accumulation, osrdr <dig> may functionally resemble the arabidopsis rdr <dig> and plays a role in the 21 nt sirna-dependent non-canonical rddm pathway  <cit> .
however, some of the analyzed loci showed increased chh methylation that is associated with increased smrna accumulation in osrdr <dig>  we should caution that because we analyzed only  <dig> loci, the results observed may not be extrapolated to global scale.
in arabidopsis, it was documented that mutation of rdr <dig> resulted in near complete loss of methylated cytosine of all three sequence contexts  within the  <dig>  chh hypomethylation dmrs  between drm1/ <dig> and wt  <cit> .
therefore, genome-wide methylation analysis  of the osrdr <dig> mutant will be required to confirm whether osrdr <dig> plays a similar role globally in rice.
previous studies in arabidopsis and nicotiana have defined an established role of rdr <dig> in plant virus responses  <cit> .
we showed here that osrdr <dig> exhibited no phenotypic differences from its sibling wt plants under normal growing condition, but displayed ephemeral phenotypic fluctuations contingent with presence of several abiotic stress conditions.
this observation, together with the enriched go categories including those involved in metabolic process of the differentially expressed genes between osrdr <dig> and wt , suggest that the effects of altered gene expression due to osrdr <dig> mutation has been largely canalized under normal condition but can be released by certain abiotic stress conditions  <cit> , an issue that merits further investigations.
regardless, our results suggest that, apart from its established role in the production and amplification of exogenous, virus-derived sirnas  in infected plants  <cit> , the rice rdr <dig> homolog  might also play a role in certain abiotic stress responses, which however may not involve stable epigenetic changes in this respect.
in this regard, it should be emphasized again that the genome-wide analyses of both smrna profiles and gene expression in osrdr <dig> were conducted on plants grown under normal conditions.
therefore, further studies are needed to conduct the analyses in plants of the mutant and wt under both short- and long-term stress conditions.
it would also be interesting to analyze the progeny of stress-treated osrdr <dig> plants to investigate if osrdr <dig> is involved in transgenerational inheritance of stress-induced epigenetic changes, if they occurred.
CONCLUSIONS
how rdr <dig> affects global gene expression and smrna profiles have not been previously investigated in any plant species.
by analyzing a null mutant of the rice rdr <dig> gene , we showed that expression of more than  <dig>  endogenous genes of diverse gene ontology  categories were significantly altered in the mutant, indicating a functional role of osrdr <dig> in regulating endogenous gene expression in rice.
by smrna deep-sequencing, we found that extensive alteration in smrna clusters occurred across each of the  <dig> rice chromosomes in the mutant, indicating a role of osrdr <dig> in smrna biogenesis and/or titration in rice.
we also found that at least some of the gene expression changes are correlated with differences in mirnas.
we further showed that changes in smrnas can be concomitant with locus-specific alteration of cytosine methylation primarily of the chh contexts, thus linking osrdr <dig> to dna methylation in rice.
finally, we showed that whereas no apparent phenotypic abnormality was associated with loss of function of osrdr <dig>  ephemeral phenotypic fluctuations could be generated by various short-term abiotic stress conditions as a result of osrdr <dig> mutation, suggesting a role of osrdr <dig> in plant abiotic stress response.

 rna silencing is an evolutionally conserved gene regulation mechanism in eukaryotes mediated by 20– nt non-coding small rnas. these smrnas are processed from double-stranded or hairpin rna molecules by dicer-like proteins, and guide rna-induced silencing complexes to cognate single-stranded rnas based on sequence complementarity, and result in degradation of the targeted rnas. in plants, there are several different classes of smrnas, including 20– nt micro rnas processed by dcl 21– nt small interfering rnas by dcl and dcl and the nt heterochromatin-associated sirnas by dcl mirnas play an important role in plant development by directing posttranscriptional gene silencing of regulatory genes such as those encoding transcription factors. similarly, 21– 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. unlike these ptgs-associated smrnas, the nt sirnas are associated with rna-directed dna methylation, a plant-specific de novo dna methylation pathway required for transcriptional silencing of transposable elements and other dna repeats to maintain genome stability. the biogenesis of sirnas in plants requires the activity of rna-dependent rna polymerase, which converts single-stranded rnas to dsrna precursors of sirnas. the dicot model plant arabidopsis thaliana has six rdr genes, i.e., rdr rdr rdr3a, rdr3b, rdr3c and rdr, of which three rdrs are shown to play roles in the rna silencing pathways. rdr is required for 24 nt sirna biogenesis and therefore involved in the canonical rddm pathway. rdr is involved in the production of the endogenous 21 nt trans-acting sirnas and also essential for sense transgene-induced ptgs. both rdr and rdr are also involved in viral sirna accumulation in infected arabidopsis plants. the function of rdr in rna silencing is less understood, but recent studies have shown that it is involved in sirna biogenesis from a subset of rna viruses. furthermore, rdr mutant of arabidopsis showed loss of dna methylation in a subset of genomic loci in comparison to wild-type arabidopsis plants, suggesting that rdr plays a role in the recently identified non-canonical, 21 nt sirna-directed rddm pathway. however, the function of rdr in gene regulation from a genome-wide perspective has not been investigated in any plant. in contrast to arabidopsis that has six rdr genes, the rdr family of rice, a model plant for monocots, contains only three members, namely osrdr osrdr and osrdr. a previous study showed that osrdr has a similar function to its counterparts in arabidopsis and tobacco in ptgs-based silencing of certain rna viruses, such as bromovirus. to investigate if osrdr plays a role in regulation of endogenous genes in rice, we characterized a loss-of-function mutant of osrdr derived from a disruptive ltr retrotransposon insertion into the 2nd exon of the gene. we investigated genome-wide changes in gene expression and smrna profiles, localized changes in dna methylation, and phenotypes under normal and several abiotic stress conditions in this rice rdr mutant.  characterization of the rice rdr mutant we obtained a ltr retrotransposon tos insertion line for osrdr from the tos insertion mutant library of rice cv. hitomebore. molecular characterization identified h as heterozygous for a tos insertion into the second exon of osrdr. we obtained the homozygous mutant and its sibling wild type plants by selfing of the heterozygous plant for five successive generations. in each generation, the three kinds of genotypes, wt, heterozygote and homozygous mutant, were selected based on locus-specific pcr amplifications. both the heterozygous and homozygous plants for osrdr showed no discernibly altered phenotypes in the entire growth and developmental period over multiple generations under normal field conditions. semi-quantitative and real-time quantitative rt-pcr analyses confirmed that the homozygous osrdr mutant had a complete loss of osrdr expression in shoot-tip tissue wherein the gene was highly expressed in wt plants. this indicated that the exonic insertion of tos knocked out the expression of osrdr and hence, abolished its function. genome-wide changes in gene expression in osrdr1 we profiled the transcriptome of shoot-tip tissues between osrdr and its sibling wt plants using the affymetrix genechip rice® genome array. after normalization of the microarray data, we detected expressed genes in the shoot-tip tissue of rice. the expression levels of genes were conserved between osrdr and wt, but and genes showed significant up- and down-regulation in osrdr respectively. a gene ontology category analysis of these differentially expressed genes showed that they were enriched in a variety of go categories. however, these differentially expressed genes were found to distribute non-randomly across the rice chromosomes. for example, chromosomes and contained significantly more distributions than the rest chromosomes. it is also clear from the data that within a given chromosome, the distribution is also nonrandom, for example, the distributions are almost exclusively confined to the long arms of chromosomes and and relative to their respective short-arms. the highly reproducible microarray profiles among three biological replicates for both osrdr and its wt sibling plants testified reliability of the data and their analysis. all microarray data have been submitted to the geo repository under the accession number of gse to further verify the quality of the microarray data and analysis, we analyzed genes representing both up- and down-regulation in osrdr vs. wt, as well as equal expression between the two lines using qrt-pcr assay on the same cdnas as used for microarray. the qrt-pcr results were highly consistent with the microarray data for almost all the tested genes in levels or at least in trends of expression changes, confirming reliability of the microarray analysis. alteration in smrna clusters in osrdr1 previous studies have established that rdr function is required for biogenesis and/or amplification of some types of rna virus-related smrna accumulation in arabidopsis and tobacco. these findings promoted us to test whether loss of function of osrdr may have a general impact on “normal” smrna abundance in rice, and we investigated this issue by high-throughput smrna sequencing. comparison of the, clean smrna reads from osrdr with the, reads from its sibling wt revealed highly similar profiles in both size distributions and sequence categories of the smrnas between osrdr and wt, suggesting that the overall smrna abundance was not generally affected by the loss of function of osrdr genome-wide overall similarity in smrna abundance does not necessitates absence of smrna fluctuations in localized smrna clusters, because up- and down-regulated smrna accumulation can be masked by reciprocal compensation. we thus investigated localized smrna accumulation between osrdr and its sibling wt by mapping the cleaned smrna reads to 100 bp sliding windows across each of the rice chromosomes, normalized the reads to reads per million, and then compared the rpm smrna clusters between osrdr and wt. using 4-fold difference as a cut-off threshold, we identified many smrna clusters with differential abundance between osrdr and its sibling wt, which were uniformly distributed across the entire length of each chromosome. next, we extracted the differentially expressed smrnas between osrdr and wt in the size range of 20-24 nt, which should parsimoniously contain all sirnas, and mapped them to the same 100 bp windows across each chromosome. we found that this subset of differential smrna clusters also distribute on both arms of each chromosome, although due to their smaller numbers, we cannot rule out the possibility that the distribution might show “hot spots” within a given chromosome. taken together, the smrna sequencing data suggested that loss-of-function mutation in osrdr caused extensive alterations in smrna clusters across each chromosome and throughout the genome, but it did not result in marked fluctuations of overall smrna profiles, probably due to more or less equally increased and decreased abundance of the smrna clusters which offset each other. altered expression of mirnas and their target genes in osrdr1 given the diverse important roles played by mirnas, we investigated if their accumulation might be affected in the osrdr mutant. previous computational and cloning studies have identified ca. mirnas from mirna families in rice. based on this information, we first analyzed the abundance of known rice mirnas in osrdr and wt. this analysis indicated that: majority of the known osa-mirnas were expressed equally or nearly so between osrdr and wt; some mirnas showed >  fold increased expression in osrdr relative to wt, with the highest expression ratio reaching:; some mirnas showed > fold decrease in expression in osrdr relative to wt, with the lowest expression ratio of 1: observed for mir167j between mutant and wt; osa-mir395p and osa-mir395s, being from the same mirna family, showed changes in expression to opposite directions, with an expression ratio of 1: for osa-mir395p but: for osa-mir395s in mutant vs. wt. to verify the expression differences based on the smrna sequencing data, we performed semi-nested qrt-pcr analysis of four mirnas in mutant and wt. the qrt-pcr results were found consistent with the smrna sequencing data, confirming the changes in mirna expression between osrdr and wt. in addition to the known mirnas, we identified a total of putative novel osa-mirnas from the smrna data of the mutant and wt plants based on prediction of pre-mirna-like stem-loop structures in sequences surrounding the smrna sequences in the rice genome. three of these novel mirnas had an identical mature sequence but corresponded to three independent genomic loci, thereby forming a novel mirna family. for the putative mirna osr-mirna-n smrna reads were detected from both the 5’ and the 3’ half of the predicted stem-loop structure, but the 5p smrna showed a higher abundance than the 3p smrna, indicating that the 5p smrna is the guide strand whereas the 3p smrna is the passenger strand. like the known mirnas, these novel mirnas also showed expression variation between osrdr and wt, with showing expression only in osrdr and showing expression only in wt plants. taken together, our results suggest that osrdr was likely involved in mirna accumulation in rice. to investigate if the altered mirna accumulation in osrdr relative to wt was associated with changes in mirna target gene expression, we compared the mirna expression profiles derived from the smrna sequencing data with the target gene expression levels based on the microarray data. we did not find a generalized relationship between the mirna abundance and target gene expression levels. instead, four types of relationships were recognized for a subset of mirnas and their predicted targets, which included: reduced mirna abundance was correlated with up-regulated expression of target genes in osrdr relative to wt; increased mirna abundance was correlated with down-regulated expression of target genes in osrdr relative to wt; both mirnas and their target genes were up-regulated in osrdr relative to wt; both mirnas and their target genes were down-regulated in osrdr relative to wt. the first two types of relationships supported a role of mirnas in down-regulating expression of their predicted target genes. the last two types of relationships could be a result of concordant transcriptional regulation of the mirnas and their target genes caused by another more upstream regulator whose expression or activity was modified due to loss of function of osrdr all small rna data have been submitted to genbank under the accession numbers of srp locus-specific alteration of dna methylation in osrdr1 as the arabidopsis rdr has been shown to play a role in the non-canonical, nerd-dependent rddm pathway, we were interested to know if osrdr might have a similar function in rice. we therefore examined cytosine methylation and gene expression levels of selected genomic loci in osrdr and its sibling wt using bisulphite sequencing and qrt-pcr analysis. these loci overlapped with two transposable elements and three protein-coding genes, which were chosen as representatives because they all showed alteration in smrna clusters in osrdr relative to wt. the bisulphite-sequenced regions for the two tes included: portions of the 5’- and 3’-ltrs together with their immediate flanking regions of two tos copies; the 5’ termini along with their immediate flanking regions of two pong copies, and; a body-region of the transposase-encoding orf of pong that is shared by all conserved copies of the element. the bisulphite-sequenced regions of the three protein-coding genes are all within their 5’-upstream regions. the bisulphite sequencing results showed that: of the five tos regions analyzed, only the 5’ ltr region for the tos copy located on chromosome showed marked decrease in cg and chg methylation but not in chh methylation in osrdr relative to wt; for the three pong regions analyzed, only the 5’region of the copy located on chromosome showed clear methylation changes: decrease in cg methylation by 20% and increase in both chg and chh methylation by approximately 30% and 50%, respectively, in osrdr relative to wt; of the three genic loci analyzed, only one showed increase in cg methylation by ca. 50% in osrdr relative to wt, while methylation of the other two regions were unchanged. the two tes showed significant up-regulation in osrdr relative to wt, consistent with a decrease in methylation at the 5’ regions of one copy of each tes in osrdr. notably, all the three genes analyzed did not show the expected relationship between dna methylation state of their 5’-regulatory regions and expression levels. specifically, one gene that showed increase in cg methylation in osrdr was up-regulated in expression; the remaining two genes showed down-regulation in osrdr relative to wt despite the lack of methylation changes in the bisulphite-sequenced regions. we next investigated possible relationships between smrna accumulation and dna methylation. we found that almost all of the altered chh methylation was associated with changes in smrna clusters. for example, the increased chh methylation of the pong copy located on chromosome was associated with a moderate increase in smrna accumulation, whereas the slight decrease in chh methylation in the flanking region and the increase in chh methylation in the gene body region of the pong copy located on chromosome were associated with moderate decrease and increase in smrna accumulations, respectively. these positive correlations of smrna accumulation and chh methylation suggests that osrdr plays a role in the de novo chh methylation in a subset of genomic loci in rice, probably by affecting the production/accumulation of smrnas required for rddm, as shown in arabidopsis. the locus-specificity of methylation changes or the two analyzed tes indicated that their methylation patterns were determined by either or both the flanking sequences and the local chromatin environment, an issue which warrants further investigations. phenotypes in osrdr under normal and abiotic stress conditions it is known that various stress conditions may produce protracted effects on genome stability, leading to transgenerational changes in genome structure, which are proposed to have been initiated by epigenetic mechanisms. we were therefore interested to know if osrdr may play a role in stress response in rice. we quantified phenotypes between osrdr and wt plants under normal and several short-term abiotic stress conditions, which included treatments with salt, heavy metals cu2+and hg2+, and overdose nitric oxide. the results showed that no phenotypic difference was found between osrdr and wt under normal condition, but significant ephemeral phenotypic differences between the two genotypes emerged in some of the different stress conditions. specifically, seedlings of osrdr were more sensitive than wt to salt and overdose no treatments, as being reflected by reduced plant height, root length and biomass at the seedling stage, with the difference in plant height and root length being persisted to the heading stage after removal of the stresses; seedlings of osrdr showed increased tolerance to heavy metal cu2+/hg2+ as indicated by increased root length, but upon removal of the stresses the differences were gradually attenuated and completely disappeared at the heading stage; when both unstressed and the transiently-stressed plants of the mutant and wt were grown to maturity, no difference in plant height, tiller number, panicle and kernel traits was observed between the two genotypes. collectively, our results suggest that osrdr has a potential function in stress response in rice, but the effects are contingent with presence of stresses without exerting protracted influence when the stresses are removed. discussion rna silencing pathways have been well characterized in the dicot model plant arabidopsis but remain poorly studied in other plants like monocots to which many major crops belong. utilizing a retransposon tos insertion mutant of osrdr in rice, we performed genome-wide analysis to unveil the function of osrdr a component recently shown in arabidopsis to be involved in non-canonical, 21 nt sirna-directed rddm pathway, on expression of endogenous genes. by deep sequencing of smrnas and microarray analysis of gene expression in the osrdr mutant and its sibling wt, we showed that the expression of >  genes were significantly changed in osrdr relative to wt, suggesting that osrdr plays a role in genome-wide gene regulation in rice. in addition, the osrdr mutant showed regional alterations in smrna accumulation and/or titration across the rice genome, and at least some of which are associated with locus-specific alteration of dna methylation. among the differentially accumulated smrnas, many were mirnas both previously known and newly identified in this study. expression changes in many of these mirnas are associated with changes in their target gene expression, which could partly be responsible for the gene expression changes observed in the osrdr mutant relative to wt. the mechanism by which mutation of osrdr caused changes in mirna expression in rice was not clear. it was found in arabidopsis that none of the rdrs has a direct role in mirna biogenesis. however, rdrs could impact mirna accumulation indirectly by either affecting mirna precursor gene expression through the tgs or ptgs pathways or generating dsrnas that compete for dcl function that is required for mirna biogenesis. our results suggested that osrdr might play a role in maintaining the intrinsic locus-specific dna methylation patterns, as its mutation caused alteration of methylation at some of the loci we analyzed. in particular, the changes in chh methylation, which are indicative of de novo methylation by the rddm pathway, showed correlation with changes in smrna accumulation. in the respect of reduced chh methylation and concomitant reduced smrna accumulation, osrdr may functionally resemble the arabidopsis rdr and plays a role in the 21 nt sirna-dependent non-canonical rddm pathway. however, some of the analyzed loci showed increased chh methylation that is associated with increased smrna accumulation in osrdr we should caution that because we analyzed only loci, the results observed may not be extrapolated to global scale. in arabidopsis, it was documented that mutation of rdr resulted in near complete loss of methylated cytosine of all three sequence contexts within the chh hypomethylation dmrs between drm1/ and wt. therefore, genome-wide methylation analysis of the osrdr mutant will be required to confirm whether osrdr plays a similar role globally in rice. previous studies in arabidopsis and nicotiana have defined an established role of rdr in plant virus responses. we showed here that osrdr exhibited no phenotypic differences from its sibling wt plants under normal growing condition, but displayed ephemeral phenotypic fluctuations contingent with presence of several abiotic stress conditions. this observation, together with the enriched go categories including those involved in metabolic process of the differentially expressed genes between osrdr and wt, suggest that the effects of altered gene expression due to osrdr mutation has been largely canalized under normal condition but can be released by certain abiotic stress conditions, an issue that merits further investigations. regardless, our results suggest that, apart from its established role in the production and amplification of exogenous, virus-derived sirnas in infected plants, the rice rdr homolog might also play a role in certain abiotic stress responses, which however may not involve stable epigenetic changes in this respect. in this regard, it should be emphasized again that the genome-wide analyses of both smrna profiles and gene expression in osrdr were conducted on plants grown under normal conditions. therefore, further studies are needed to conduct the analyses in plants of the mutant and wt under both short- and long-term stress conditions. it would also be interesting to analyze the progeny of stress-treated osrdr plants to investigate if osrdr is involved in transgenerational inheritance of stress-induced epigenetic changes, if they occurred.  how rdr affects global gene expression and smrna profiles have not been previously investigated in any plant species. by analyzing a null mutant of the rice rdr gene, we showed that expression of more than endogenous genes of diverse gene ontology categories were significantly altered in the mutant, indicating a functional role of osrdr in regulating endogenous gene expression in rice. by smrna deep-sequencing, we found that extensive alteration in smrna clusters occurred across each of the rice chromosomes in the mutant, indicating a role of osrdr in smrna biogenesis and/or titration in rice. we also found that at least some of the gene expression changes are correlated with differences in mirnas. we further showed that changes in smrnas can be concomitant with locus-specific alteration of cytosine methylation primarily of the chh contexts, thus linking osrdr to dna methylation in rice. finally, we showed that whereas no apparent phenotypic abnormality was associated with loss of function of osrdr ephemeral phenotypic fluctuations could be generated by various short-term abiotic stress conditions as a result of osrdr mutation, suggesting a role of osrdr in plant abiotic stress response. 
recent studies have suggested that rdr 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. it has been established that rdr is involved in the 24 nt sirna-dependent rna-directed dna methylation pathway. some of the regions with altered smrna clusters were associated with changes in dna methylation. our results showed that osrdr 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. gene expressionepigeneticssmall rnadna methylationrdr1oryza sativa l despite these changes, no phenotypic difference was identified in osrdr relative to wt under normal condition; however, ephemeral phenotypic fluctuations occurred under some abiotic stress conditions. microarray analysis identified differentially expressed genes between osrdr and wt, of which and genes were up- and down-regulated, respectively, in osrdr smrna sequencing revealed regional alterations in smrna clusters across the rice genome. we characterized a loss-of-function mutant of the osrdr gene in rice derived from a retrotransposon tos insertion.
 endogenous small rnas are important trans/cis-acting regulators involved in diverse cellular functions. in plants, the rna-dependent rna polymerases are essential for smrna biogenesis. it has been established that rdr is involved in the 24 nt sirna-dependent rna-directed dna methylation pathway. recent studies have suggested that rdr 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 rdr may have similar functions in other plants remained unknown.  we characterized a loss-of-function mutant of the osrdr gene in rice derived from a retrotransposon tos insertion. microarray analysis identified differentially expressed genes between osrdr and wt, of which and genes were up- and down-regulated, respectively, in osrdr 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 osrdr 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 osrdr relative to wt under normal condition; however, ephemeral phenotypic fluctuations occurred under some abiotic stress conditions.  our results showed that osrdr 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. gene expressionepigeneticssmall rnadna methylationrdr1oryza sativa l 