Evolutionary genomics of two diploid goat grass species belonging to the section Sitopsis of Aegilops, Aegilops longissima, and Aegilops sharonensis.

Aegilops longissima and Ae. sharonensis, being classified into the Sitopsis section of genus Aegilops, are distinct species both taxonomically and ecologically. Nevertheless, earlier observations indicate that the two species are not reproductively isolated to full extent and can inter-bred upon secondary contact. However, the genomic underpinnings of the morpho-ecological differentiation between the two foci species remained unexplored. Here, we resequenced 31 representative accessions of the two species and conducted in-depth comparative genomic analyses. We demonstrate recurrent and ongoing natural hybridizations between Ae. longissima and Ae. sharonensis, and depict features of genome composition of the resultant hybrids at both individual and population levels. We also delineate genomic regions and candidate genes potentially underpinning the differential morphological and edaphic adaptations of the two species. Intriguingly, a binary morphology was observed in the hybrids, suggesting existence of highly diverged genomic regions that remain uneroded by the admixtures. Together, our results provide new insights into the molding effects of interspecific hybridization on genome composition and mechanisms preventing merge of the two species.

Effects of Allopolyploidization and Homoeologous Chromosomal Segment Exchange on Homoeolog Expression in a Synthetic Allotetraploid Wheat under Variable Environmental Conditions.

Allopolyploidy through the combination of divergent genomes into a common nucleus at doubled dosage is known as a potent genetic and evolutionary force. As a macromutation, a striking feature of allopolyploidy in comparison with other mutational processes is that 'genome shock' can be evoked, thereby generating rapid and saltational biological consequences. A major manifestation of genome shock is genome-wide gene expression rewiring, which previously remained to be fully elucidated. Here, using a large set of RNAseq-based transcriptomic data of a synthetic allotetraploid wheat (genome AADD) and its parental species, we performed in-depth analyses of changes in the genome-wide gene expression under diverse environmental conditions at the subgenome (homoeolog) level and investigated the additional effects of homoeologous chromosomal segment exchanges (abbreviated HEs). We show that allopolyploidy caused large-scale changes in gene expression that were variable across the conditions and exacerbated by both stresses and HEs. Moreover, although both subgenomes (A and D) showed clear commonality in the changes, they responded differentially under variable conditions. The subgenome- and condition-dependent differentially expressed genes were enriched for different gene ontology terms implicating different biological functions. Our results provide new insights into the direct impacts of allopolyploidy on condition-dependent changes in subgenome expression and the additional effects of HEs in nascent allopolyploidy.

Meiotic pairing irregularity and homoeologous chromosome compensation cause rapid karyotype variation in synthetic allotetraploid wheat.

Allopolyploidization may initiate rapid evolution due to heritable karyotypic changes. The types and extents of these changes, the underlying causes, and their effects on phenotype remain to be fully understood. Here, we designed experimental populations suitable to address these issues using a synthetic allotetraploid wheat. We show that extensive variation in both chromosome number (NCV) and structure (SCV) accumulated in a selfed population of a synthetic allotetraploid wheat (genome Sb Sb DD). The combination of NCVs and SCVs generated massive organismal karyotypic heterogeneity. NCVs and SCVs were intrinsically correlated and highly variable across the seven sets of homoeologous chromosomes. Both NCVs and SCVs stemmed from meiotic pairing irregularity (presumably homoeologous pairing) but were also constrained by homoeologous chromosome compensation. We further show that homoeologous meiotic pairing was positively correlated with sequence synteny at the subtelomeric regions of both chromosome arms, but not with genic nucleotide similarity per se. Both NCVs and SCVs impacted phenotypic traits but only NCVs caused significant reduction in reproductive fitness. Our results implicate factors influencing meiotic homoeologous chromosome pairing and reveal the type and extent of karyotypic variation and its immediate phenotypic manifestation in synthetic allotetraploid wheat. This has relevance for our understanding of allopolyploid evolution.

Overexpression of a Cinnamyl Alcohol Dehydrogenase-Coding Gene, GsCAD1, from Wild Soybean Enhances Resistance to Soybean Mosaic Virus.

Soybean mosaic virus (SMV) is the most prevalent soybean viral disease in the world. As a critical enzyme in the secondary metabolism of plants, especially in lignin synthesis, cinnamyl alcohol dehydrogenase (CAD) is widely involved in plant growth and development, and in defense against pathogen infestation. Here, we performed RNAseq-based transcriptome analyses of a highly SMV-resistant accession (BYO-15) of wild soybean (Glycine soja) and a SMV-susceptible soybean cultivar (Williams 82), also sequenced together with a resistant plant and a susceptible plant of their hybrid descendants at the F3 generation at 7 and 14 days post-inoculation with SMV. We found that the expression of GsCAD1 (from G. soja) was significantly up-regulated in the wild soybean and the resistant F3 plant, while the GmCAD1 from the cultivated soybean (G. max) did not show a significant and persistent induction in the soybean cultivar and the susceptible F3 plant, suggesting that GsCAD1 might play an important role in SMV resistance. We cloned GsCAD1 and overexpressed it in the SMV-susceptible cultivar Williams 82, and we found that two independent GsCAD1-overexpression (OE) lines showed significantly enhanced SMV resistance compared with the non-transformed wild-type (WT) control. Intriguingly, the lignin contents in both OE lines were higher than the WT control. Further liquid chromatography (HPLC) analysis showed that the contents of salicylic acid (SA) were significantly more improved in the OE lines than that of the wild-type (WT), coinciding with the up-regulated expression of an SA marker gene. Finally, we observed that GsCAD1-overexpression affected the accumulation of SMV in leaves. Collectively, our results suggest that GsCAD1 enhances resistance to SMV in soybeans, most likely by affecting the contents of lignin and SA.

Homoeologous exchange enables rapid evolution of tolerance to salinity and hyper-osmotic stresses in a synthetic allotetraploid wheat.

The link between polyploidy and enhanced adaptation to environmental stresses could be a result of polyploidy itself harbouring higher tolerance to adverse conditions, or polyploidy possessing higher evolvability than diploids under stress conditions. Natural polyploids are inherently unsuitable to disentangle these two possibilities. Using selfed progenies of a synthetic allotetraploid wheat AT3 (AADD) along with its diploid parents, Triticum urartu TMU38 (AA) and Aegilops tauschii TQ27 (DD), we addressed the foregoing issue under abiotic salinity and hyper-osmotic (drought-like) stress. Under short duration of both stresses, euploid plants of AT3 showed intermediate tolerance of diploid parents; under life-long duration of both stresses, tolerant individuals to either stress emerged from selfed progenies of AT3, but not from comparable-sized diploid parent populations. Tolerance to both stresses were conditioned by the same two homoeologous exchanges (HEs; 2DS/2AS and 3DL/3AL), and at least one HE needed to be at the homozygous state. Transcriptomic analyses revealed that hyper-up-regulation of within-HE stress responsive genes of the A sub-genome origin is likely responsible for the dual-stress tolerant phenotypes. Our results suggest that HE-mediated inter-sub-genome rearrangements can be an important mechanism leading to adaptive evolution in allopolyploids as well as a promising target for genetic manipulation in crop improvement.

Effects of homoeologous exchange on gene expression and alternative splicing in a newly formed allotetraploid wheat.

Homoeologous exchange (HE) is a major mechanism generating post-polyploidization genetic variation with important evolutionary consequences. However, the direct impacts of HE on gene expression and transcript diversity in allopolyploids without the intertwined evolutionary processes remain to be fully understood. Here, we analyzed high-throughput RNA-seq data of young leaves from plant groups of a synthetic allotetraploid wheat (AADD), which contained variable numbers of HEs. We aimed to investigate if and to which extent HE directly impacts gene expression and alternative splicing (AS). We found that HE impacts expression of genes located within HE regions primarily via a cis-acting dosage effect, which led to significant changes in the total expression level of homoeologous gene pairs, especially for homoeologs whose original expression was biased. In parallel, HE also influences expression of a large number of genes residing in non-HE regions by trans-regulation leading to convergent expression of homoeologs. Intriguingly, when taking the original relative homoeolog expression states into account, homoeolog pairs under trans-effect are more prone to manifesting a convergent response to the HEs whereas those under cis-regulation tended to show further exacerbated subgenome-biased expression. Moreover, HE-induced quantitative, largely individual-specific, changes of AS events were detected. Similar to homoeologous expression, homoeo-AS events under trans-effect were more responsive to HE. HE therefore exerts multifaceted immediate effects on gene expression and, to a less extent, on individualized transcript diversity in nascent allopolyploidy.

Mutation of GmAITR Genes by CRISPR/Cas9 Genome Editing Results in Enhanced Salinity Stress Tolerance in Soybean.

Breeding of stress-tolerant plants is able to improve crop yield under stress conditions, whereas CRISPR/Cas9 genome editing has been shown to be an efficient way for molecular breeding to improve agronomic traits including stress tolerance in crops. However, genes can be targeted for genome editing to enhance crop abiotic stress tolerance remained largely unidentified. We have previously identified abscisic acid (ABA)-induced transcription repressors (AITRs) as a novel family of transcription factors that are involved in the regulation of ABA signaling, and we found that knockout of the entire family of AITR genes in Arabidopsis enhanced drought and salinity tolerance without fitness costs. Considering that AITRs are conserved in angiosperms, AITRs in crops may be targeted for genome editing to improve abiotic stress tolerance. We report here that mutation of GmAITR genes by CRISPR/Cas9 genome editing leads to enhanced salinity tolerance in soybean. By using quantitative RT-PCR analysis, we found that the expression levels of GmAITRs were increased in response to ABA and salt treatments. Transfection assays in soybean protoplasts show that GmAITRs are nucleus proteins, and have transcriptional repression activities. By using CRISPR/Cas9 to target the six GmAITRs simultaneously, we successfully generated Cas9-free gmaitr36 double and gmaitr23456 quintuple mutants. We found that ABA sensitivity in these mutants was increased. Consistent with this, ABA responses of some ABA signaling key regulator genes in the gmaitr mutants were altered. In both seed germination and seedling growth assays, the gmaitr mutants showed enhanced salt tolerance. Most importantly, enhanced salinity tolerance in the mutant plants was also observed in the field experiments. These results suggest that mutation of GmAITR genes by CRISPR/Cas9 is an efficient way to improve salinity tolerance in soybean.

Analysis of expression characteristics of soybean leaf and root tissue-specific promoters in Arabidopsis and soybean.

The characterization of tissue-specific promoters is critical for studying the functions of genes in a given tissue/organ. To study tissue-specific promoters in soybean, we screened tissue-specific expressed genes using published soybean RNA-Seq-based transcriptome data coupled with RT-PCR analysis. We cloned the promoters of three genes, GmADR1, GmBTP1, and GmGER1, and constructed their corresponding ß-Glucuronidase (GUS) promoter-GUS reporter vectors. We generated transgenic Arabidopsis plants and examined the expression patterns of these promoters by GUS staining and RT-PCR analysis. We also transformed the promoter-GUS reporter vectors into soybean to obtain hairy roots, and examined promoter expression by GUS staining. We found a root-specific expression pattern of GmADR1 and GmBTP1 in both Arabidopsis and soybean, and the promoter of GmGER1 showed a leaf-specific pattern in transgenic Arabidopsis plants. To test the potential utility of these promoters in soybean improvement by transgenic means, we used the GmADR1 promoter to drive expression of a salt resistance gene in soybean, GmCaM4, by generating transgenic soybean plants. We found that the transgenic plants had significantly enhanced salt tolerance compared to non-transformed wild-type, suggesting that introducing endogenous promoters by transgenic means can drive the expression of functional genes in specific tissues and organs in soybean.

Elastin-like polypeptide and γ-zein fusions significantly increase recombinant protein accumulation in soybean seeds.

Soybean seeds are an ideal host for the production of recombinant proteins because of their high content of proteins, long-term stability of seed proteins under ambient conditions, and easy establishment of efficient purification protocols. In this study, a polypeptide fusion strategy was applied to explore the capacity of elastin-like polypeptide (ELP) and γ-zein fusions in increasing the accumulation of the recombinant protein in soybean seeds. Transgenic soybean plants were generated to express the γ-zein- or ELP-fused green fluorescent protein (GFP) under the control of the soybean seed-specific promoter of ß-conglycinin alpha subunit (BCSP). Significant differences were observed in the accumulation of zein-GFP and GFP-ELP from that of the unfused GFP in transgenic soybean seeds based on the total soluble protein (TSP), despite the low-copy of T-DNA insertions and similar expression at the mRNA levels in selected transgenic lines. The average levels of zein-GFP and GFP-ELP accumulated in immature seeds of these transgenic lines were 0.99% and 0.29% TSP, respectively, compared with 0.07% TSP of the unfused GFP. In mature soybean seeds, the accumulation of zein-GFP and GFP-ELP proteins was 1.8% and 0.84% TSP, an increase of 3.91- and 1.82-fold, respectively, in comparison with that of the unfused GFP (0.46% TSP). Confocal laser scanning analysis showed that both zein-GFP and GFP-ELP were abundantly deposited in many small spherical particles of transgenic seeds, while there were fewer such florescence signals in the same cellular compartments of the unfused GFP-expressing seeds. Despite increased recombinant protein accumulation, there were no significant changes in the total protein and oil content in seeds between the transgenic and non-transformed plants, suggesting the possible presence of threshold limits of total protein accumulation in transgenic soybean seeds. Overall, our results indicate that γ-zein and ELP fusions significantly increased the accumulation of the recombinant protein, but exhibited no significant influence on the total protein and oil content in soybean seeds.

MYB transcription factors GmMYBA2 and GmMYBR function in a feedback loop to control pigmentation of seed coat in soybean.

Soybean has undergone extensive selection pressures for seed nutrient composition and seed color during domestication, but the major genetic loci controlling seed coat color have not been completely understood, and the transcriptional regulation relationship among the loci remains elusive. Here, two major regulators, GmMYBA2 and GmMYBR, were functionally characterized as an anthocyanin activator and repressor, respectively. Ectopic expression of GmMYBA2 in soybean hairy roots conferred the enhanced accumulation of delphinidin and cyanidin types of anthocyanins in W1t and w1T backgrounds, respectively, through activating anthocyanin biosynthetic genes in the reported loci. The seed coat pigmentation of GmMYBA2-overexpressing transgenic plants in the W1 background mimicked the imperfect black phenotype (W1/w1, i, R, t), suggesting that GmMYBA2 was responsible for the R locus. Molecular and biochemical analysis showed that GmMYBA2 interacted with GmTT8a to directly activate anthocyanin biosynthetic genes. GmMYBA2 and GmMYBR might form a feedback loop to fine-tune seed coat coloration, which was confirmed in transgenic soybeans. Both GmTT8a and GmMYBR that were activated by GmMYBA2 in turn enhanced and obstructed the formation of the GmMYBA2-GmTT8a module, respectively. The results revealed the sophisticated regulatory network underlying the soybean seed coat pigmentation loci and shed light on the understanding of the seed coat coloration and other seed inclusions.

Mutation of a major CG methylase alters genome-wide lncRNA expression in rice.

Plant long non-coding RNAs (lncRNAs) function in diverse biological processes, and lncRNA expression is under epigenetic regulation, including by cytosine DNA methylation. However, it remains unclear whether 5-methylcytosine (5mC) plays a similar role in different sequence contexts (CG, CHG, and CHH). In this study, we characterized and compared the profiles of genome-wide lncRNA profiles (including long intergenic non-coding RNAs [lincRNAs] and long noncoding natural antisense transcripts [lncNATs]) of a null mutant of the rice DNA methyltransferase 1, OsMET1-2 (designated OsMET1-2-/-) and its isogenic wild type (OsMET1-2+/+). The En/Spm transposable element (TE) family, which was heavily methylated in OsMET1-2+/+, was transcriptionally de-repressed in OsMET1-2-/- due to genome-wide erasure of CG methylation, and this led to abundant production of specific lncRNAs. In addition, RdDM-mediated CHH hypermethylation was increased in the 5'-upstream genomic regions of lncRNAs in OsMET1-2-/-. The positive correlation between the expression of lincRNAs and that of their proximal protein-coding genes was also analyzed. Our study shows that CG methylation negatively regulates the TE-related expression of lncRNA and demonstrates that CHH methylation is also involved in the regulation of lncRNA expression.

Homoeologous exchanges occur through intragenic recombination generating novel transcripts and proteins in wheat and other polyploids.

Recombination between homeologous chromosomes, also known as homeologous exchange (HE), plays a significant role in shaping genome structure and gene expression in interspecific hybrids and allopolyploids of several plant species. However, the molecular mechanisms that govern HEs are not well understood. Here, we studied HE events in the progeny of a nascent allotetraploid (genome AADD) derived from two diploid progenitors of hexaploid bread wheat using cytological and whole-genome sequence analyses. In total, 37 HEs were identified and HE junctions were mapped precisely. HEs exhibit typical patterns of homologous recombination hotspots, being biased toward low-copy, subtelomeric regions of chromosome arms and showing association with known recombination hotspot motifs. But, strikingly, while homologous recombination preferentially takes place upstream and downstream of coding regions, HEs are highly enriched within gene bodies, giving rise to novel recombinant transcripts, which in turn are predicted to generate new protein fusion variants. To test whether this is a widespread phenomenon, a dataset of high-resolution HE junctions was analyzed for allopolyploid Brassica, rice, Arabidopsis suecica, banana, and peanut. Intragenic recombination and formation of chimeric genes was detected in HEs of all species and was prominent in most of them. HE thus provides a mechanism for evolutionary novelty in transcript and protein sequences in nascent allopolyploids.

Transcriptome Changes during Major Developmental Transitions Accompanied with Little Alteration of DNA Methylome in Two Pleurotus Species.

Pleurotus tuoliensis (Pt) and P. eryngii var. eryngii (Pe) are important edible mushrooms. The epigenetic and gene expression signatures characterizing major developmental transitions in these two mushrooms remain largely unknown. Here, we report global analyses of DNA methylation and gene expression in both mushrooms across three major developmental transitions, from mycelium to primordium and to fruit body, by whole-genome bisulfite sequencing (WGBS) and RNA-seq-based transcriptome profiling. Our results revealed that in both Pt and Pe the landscapes of methylome are largely stable irrespective of genomic features, e.g., in both protein-coding genes and transposable elements (TEs), across the developmental transitions. The repressive impact of DNA methylation on expression of a small subset of genes is likely due to TE-associated effects rather than their own developmental dynamics. Global expression of gene orthologs was also broadly conserved between Pt and Pe, but discernible interspecific differences exist especially at the fruit body formation stage, and which are primarily due to differences in trans-acting factors. The methylome and transcriptome repertories we established for the two mushroom species may facilitate further studies of the epigenetic and transcriptional regulatory mechanisms underpinning gene during development in Pleurotus and related genera.

Extensive changes in gene expression and alternative splicing due to homoeologous exchange in rice segmental allopolyploids.

KEY MESSAGE: We report rampant homoeologous exchanges in progenies of a newly synthesized rice segmental allotetraploid and demonstrate their consequences to changes of gene expression and alternative splicing. Allopolyploidization is recurrent across the tree of angiosperms and known as a driving evolutionary force in both plants and animals. A salient feature of allopolyploidization is the induction of homoeologous exchange (HE) events between the constituent subgenomes, which may in turn cause changes in gene expression, transcript alternative splicing, and phenotypic novelty. However, this issue has been poorly studied, largely because lack of a system in which the exact parentage donating the subgenomes is known and the HE events are occurring in real time. Here, we employed whole-genome re-sequencing and RNA-seq-based transcriptome profiling in four randomly chosen progeny individuals (at the 10th-selfed generation) of segmental allotetraploids that were constructed by colchicine-mediated whole-genome doubling of F1 hybrids between the two subspecies (japonica and indica) of Asian cultivated Oryza sativa. We show that rampant HE events occurred in these tetraploid individuals, which converted most of the otherwise heterozygous genomic regions into a homogenized state of one parental subgenome. We demonstrate that genes within these homogenized genomic regions in the tetraploids showed high frequencies of altered expression and enhanced alternative splicing relative to their counterparts in the corresponding diploid parents in the embryo tissue. Intriguingly, limited overlaps between the differentially expressed genes and the differential alternative spliced genes were identified, which were partitioned to distinctly enriched gene ontology terms. Together, our results indicate that HE is a major mechanism to rapidly generate novelty in gene expression and transcriptome diversity, which may facilitate phenotypic innovation in nascent allopolyploids and relevant to allopolyploid crop breeding.

Over-expression of GmKR3, a TIR-NBS-LRR type R gene, confers resistance to multiple viruses in soybean.

KEY MESSAGE: That overexpression of GmKR3 enhances innate virus resistance by stimulating. Soybean mosaic virus (SMV) is found in many soybean production areas, and SMV infection is one of the prevalent viral diseases that can cause significant yield losses in soybean. In plants, resistance (R) genes are involved in pathogen reorganization and innate immune response activation. Most R proteins have nucleotide-binding site and leucine-rich repeat (NBS-LRR) domains, and some of the NBS-LRR type R proteins in dicots have Toll/Interleukin-1 Receptor (TIR) motifs. We report here the analysis of the over-expression of GmKR3, a soybean TIR-NBS-LRR type R gene on virus resistance in soybean. When over-expressed in soybean, GmKR3 enhanced the plant's resistance to several strains of SMV, the closely related potyviruses bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV), and the secovirus bean pod mottle virus (BPMV). Importantly, over-expression of GmKR3 did not affect plant growth and development, including yield and qualities of the seeds. HPLC analysis showed that abscisic acid (ABA) content increased in the 35S:GmKR3 transgenic plants, and in both wild-type and 35S:GmKR3 transgenic plants in response to virus inoculation. Consistent with this observation, we found that the expression of two ABA catabolism genes was down-regulated in 35S:GmKR3 transgenic plants. We also found that the expression of Gm04.3, an ABA responsive gene encoding BURP domain-containing protein, was up-regulated in 35S:GmKR3 transgenic plants. Taken together, our results suggest that overexpression of GmKR3 enhanced virus resistance in soybean, which was achieved at least in part via ABA signaling.

Zinc oxide nanoparticle exposure triggers different gene expression patterns in maize shoots and roots.

The potential impacts of environmentally accumulated zinc oxide nanoparticles (nZnOs) on plant growth have not been well studied. A transcriptome profile analysis of maize exposed to nZnOs showed that the genes in the shoots and roots responded differently. Although the number of differentially expressed genes (DEGs) in the roots was greater than that in the shoots, the number of up- or down-regulated genes in both the shoots and roots was similar. The enrichment of gene ontology (GO) terms was also significantly different in the shoots and roots. The "nitrogen compound metabolism" and "cellular component" terms were specifically and highly up-regulated in the nZnO-exposed roots, whereas the categories "cellular metabolic process", "primary metabolic process" and "secondary metabolic process" were down-regulated in the exposed roots only. Our results revealed the DEG response patterns in maize shoots and roots after nZnO exposure.

Over-expression of GmSN1 enhances virus resistance in Arabidopsis and soybean.

KEY MESSAGE: GmSN1 enhances virus resistance in plants most likely by affecting the expression of signal transduction and immune response genes. Soybean mosaic virus (SMV) infection causes severe symptom and leads to massive yield loss in soybean (Glycine max). By comparative analyzing gene expression in the SMV-resistant soybean cultivar Rsmv1 and the susceptible cultivar Ssmv1 at a transcriptome level, we found that a subgroup of Gibberellic Acid Stimulated Transcript (GAST) genes were down-regulated in SMV inoculated Ssmv1 plants, but not Rsmv1 plants. Sequence alignment and phylogenetic analysis indicated that one of the GAST genes, GmSN1, was closely related to Snakin-1, a well-characterized potato microbial disease resistance gene. When over-expressed in Arabidopsis and soybean, respectively, under the control of the 35S promoter, GmSN1 enhanced turnip mosaic virus resistance in the transgenic Arabidopsis plants, and SMV resistance in the transgenic soybean plants, respectively. Transcriptome analysis results showed that the up-regulated genes in the 35S:GmSN1 transgenic Arabidopsis plants were largely enriched in functional terms including "signal transduction" and "immune response". Real-time PCR assay indicated that the expression of GmAKT2, a potassium channel gene known to enhance SMV resistance when over-expressed in soybean, was elevated in the 35S:GmSN1 transgenic soybean plants. Taken together, our results suggest that GmSN1 enhances virus resistance in plants most likely by affecting the expression of signal transduction and immune response genes.

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.

BACKGROUND: Endogenous 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. RESULTS: We 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. CONCLUSIONS: Our 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.