Characterization of unique small RNA populations from rice grain

Small RNAs (approximately 20 to 24 nucleotides) function as naturally occurring molecules critical in developmental pathways in plants and animals. Here we analyze small RNA populations from mature rice grain and seedlings by pyrosequencing. Using a clustering algorithm to locate regions producing small RNAs, we classified hotspots of small RNA generation within the genome. Hotspots here are defined as 1 kb regions within which small RNAs are significantly overproduced relative to the rest of the genome. Hotspots were identified to facilitate characterization of different categories of small RNA regulatory elements. Included in the hotspots, we found known members of 23 miRNA families representing 92 genes, one trans acting siRNA (ta-siRNA) gene, novel siRNA-generating coding genes and phased siRNA generating genes. Interestingly, over 20% of the small RNA population in grain came from a single foldback structure, which generated eight phased 21-nt siRNAs. This is reminiscent of a newly arising miRNA derived from duplication of progenitor genes. Our results provide data identifying distinct populations of small RNAs, including phased small RNAs, in mature grain to facilitate characterization of small regulatory RNA expression in monocot species.     

Viral infection induces expression of novel phased microRNAs from conserved cellular microRNA precursors

RNA silencing, mediated by small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs), is a potent antiviral or antibacterial mechanism, besides regulating normal cellular gene expression critical for development and physiology. To gain insights into host small RNA metabolism under infections by different viruses, we used Solexa/Illumina deep sequencing to characterize the small RNA profiles of rice plants infected by two distinct viruses, Rice dwarf virus (RDV, dsRNA virus) and Rice stripe virus (RSV, a negative sense and ambisense RNA virus), respectively, as compared with those from non-infected plants. Our analyses showed that RSV infection enhanced the accumulation of some rice miRNA*s, but not their corresponding miRNAs, as well as accumulation of phased siRNAs from a particular precursor. Furthermore, RSV infection also induced the expression of novel miRNAs in a phased pattern from several conserved miRNA precursors. In comparison, no such changes in host small RNA expression was observed in RDV-infected rice plants. Significantly RSV infection elevated the expression levels of selective OsDCLs and OsAGOs, whereas RDV infection only affected the expression of certain OsRDRs. Our results provide a comparative analysis, via deep sequencing, of changes in the small RNA profiles and in the genes of RNA silencing machinery induced by different viruses in a natural and economically important crop host plant. They uncover new mechanisms and complexity of virus-host interactions that may have important implications for further studies on the evolution of cellular small RNA biogenesis that impact pathogen infection, pathogenesis, as well as organismal development.     

Distinct Regulation of Adaxial-Abaxial Polarity in Anther Patterning in Rice

Establishment of adaxial-abaxial polarity is essential for lateral organ development. The mechanisms underlying the polarity establishment in the stamen remain unclear, whereas those in the leaf are well understood. Here, we investigated a rod-like lemma (rol) mutant of rice (Oryza sativa), in which the development of the stamen and lemma is severely compromised. We found that the rod-like structure of the lemma and disturbed anther patterning resulted from defects in the regulation of adaxial-abaxial polarity. Gene isolation indicated that the rol phenotype was caused by a weak mutation in SHOOTLESS2 (SHL2), which encodes an RNA-dependent RNA polymerase and functions in trans-acting small interfering RNA (ta-siRNA) production. Thus, ta-siRNA likely plays an important role in regulating the adaxial-abaxial polarity of floral organs in rice. Furthermore, we found that the spatial expression patterns of marker genes for adaxial-abaxial polarity are rearranged during anther development in the wild type. After this rearrangement, a newly formed polarity is likely to be established in a new developmental unit, the theca primordium. This idea is supported by observations of abnormal stamen development in the shl2-rol mutant. By contrast, the stamen filament is likely formed by abaxialization. Thus, a unique regulatory mechanism may be involved in regulating adaxial-abaxial polarity in stamen development.     

The REDUCED LEAFLET Genes Encode Key Components of the trans-Acting Small Interfering RNA Pathway and Regulate Compound Leaf and Flower Development in Lotus japonicus

The endogenous trans-acting small interfering RNA (ta-siRNA) pathway plays a conserved role in adaxial-abaxial patterning of lateral organs in simple-leafed plant species. However, its function in compound-leafed species is largely unknown. Using the compound-leafed species Lotus japonicus, we identified and characterized two independent mutants, reduced leaflet1 (rel1) and rel3, whose most conspicuous defects in compound leaves are abaxialized leaflets and reduction in leaflet number. Concurrent mutations in REL genes also compromise flower development and result in radial symmetric floral organs. Positional cloning revealed that REL1 and REL3 encode the homologs of Arabidopsis (Arabidopsis thaliana) SUPPRESSOR OF GENE SILENCING3 and ARGONAUTE7/ZIPPY, respectively, which are key components of the ta-siRNA pathway. These observations, together with the expression and functional data, demonstrated that the ta-siRNA pathway plays conserved yet distinct roles in the control of compound leaf and flower development in L. japonicus. Moreover, the phenotypic alterations of lateral organs in ta-siRNA-deficient mutants and the regulation of downstream targets by the ta-siRNA pathway in L. japonicus were similar to those in the monocots but different from Arabidopsis, indicating many parallels between L. japonicus and the monocots in the control of lateral organ development by the ta-siRNA pathway.Plant endogenous small RNAs can be categorized into microRNAs (miRNAs) and small interfering RNAs (siRNAs) according to their mechanism of biogenesis (Vaucheret, 2006). trans-Acting siRNAs (ta-siRNAs) are one type of siRNA, and their biogenesis requires several key components, such as SUPPRESSOR OF GENE SILENCING3 (SGS3), RNA-DEPENDENT RNA POLYMERASE6 (RDR6), DICER-LIKE4 (DCL4), ARGONAUTE7 (AGO7)/ZIPPY (ZIP), and dsRNA-BINDING4 (Peragine et al., 2004; Vazquez et al., 2004; Gasciolli et al., 2005; Xie et al., 2005; Yoshikawa et al., 2005; Adenot et al., 2006; Nakazawa et al., 2007). Recent studies revealed that the ta-siRNA pathway is integrated into different processes of plant development, such as vegetative phase transition in Arabidopsis (Arabidopsis thaliana; Hunter et al., 2003; Peragine et al., 2004; Xie et al., 2005; Nakazawa et al., 2007) and shoot apical meristem (SAM) initiation in rice (Oryza sativa; Satoh et al., 1999; Itoh et al., 2000; Nagasaki et al., 2007). Parallel studies of this pathway in simple-leafed species also showed that the ta-siRNA pathway plays critical roles in patterning of leaves and floral organs.In flowering plants, leaves and flowers are produced on the periphery of the apical meristem. These lateral organs are structurally asymmetric with regard to the apical meristem. The adaxial side is adjacent to the meristem, while the abaxial side is away from the meristem. The ta-siRNA pathway was found to play a conserved role in specifying the adaxial identity of lateral organs in both monocots and dicots, but defects in the ta-siRNA pathway caused more severe phenotypes in monocots than in dicot Arabidopsis. In Arabidopsis, no clear leaf polarity defects were detected in the ta-siRNA-defective mutants. However, blocking the ta-siRNA pathway in asymmetric1 (as1) or as2 background, which are regulators of leaf adaxial identity (Lin et al., 2003; Xu et al., 2003), results in enhanced adaxial-abaxial leaf defects (Li et al., 2005; Xu et al., 2006; Garcia et al., 2006). In addition, the as2rdr6 double mutants also display aberrant flowers with sepals failing to enwrap the inner whorl organs and some sepals and petals becoming needle-like structures (Li et al., 2005). In maize (Zea mays), mutations in LEAFBLADELESS1 (LBL1), which encodes the Arabidopsis SGS3 ortholog, give rise to abnormal leaves with partial or complete loss of adaxial cell identity (Timmermans et al., 1998; Nogueira et al., 2007). In severe lbl1 mutants, leaf-like lateral organs of inflorescences and flowers develop as symmetric, thread-like organs, and the immature ear is exposed and arrested in development (Timmermans et al., 1998). In rice, the osdcl4-1 mutants display an abaxialized epidermis in coleoptiles and in the first leaf, and knockdown of OsDCL4 can lead to the awn-like lemma with a radial abaxialized identity and the stamens and carpel not enwrapped by the lemma and pelea (Liu et al., 2007). Transgenic rice plants with ectopic expression of SHOOTLESS4 (SHL4), the homolog of Arabidopsis AGO7, exhibit partially adaxialized leaves (Nagasaki et al., 2007; Shi et al., 2007).In addition to the ta-siRNA pathway, other components have also been shown to be involved in the adaxial-abaxial patterning of lateral organs. The Antirrhinum majus PHANTASTICA (PHAN) gene (Waites et al., 1998; Byrne et al., 2000; Xu et al., 2003; Qi et al., 2004), which is the ortholog of Arabidopsis AS1, and CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) gene family members (McConnell et al., 2001; Emery et al., 2003) contribute to adaxial pattern formation of lateral organs, whereas members of YABBY (YAB; Sawa et al., 1999; Siegfried et al., 1999) and KANADI (Eshed et al., 2001; Kerstetter et al., 2001) gene families, AUXIN RESPONSE FACTOR3 (ARF3) and ARF4 (Pekker et al., 2005), and the miRNAs miR165/166 (Emery et al., 2003; Eshed et al., 2004; Mallory et al., 2004) are required for specifying abaxial identity. How the activities of these adaxial and abaxial determinants are coordinated has been extensively studied. It was found that ARF3 and ARF4 are regulated by the TAS3 ta-siRNA, and this regulation is conserved in both monocots and dicots (Allen et al., 2005; Williams et al., 2005). Recent studies in Arabidopsis suggest that ta-siRNAs act in a non-cell-autonomous manner to spatially restrict ARF activity (Chitwood et al., 2009; Schwab et al., 2009).In contrast to simple leaves with their single lamina, compound leaves are composed of one petiole and several leaflets. It is found that genes required for the adaxial-abaxial patterning of lateral organs in simple-leafed species also play critical roles in compound-leafed species, but these genes play multiple roles in compound leaf development. In tomato (Solanum lycopersicum), down-regulation of PHAN ortholog disturbs the leaf polarity as well as leaflet formation (Kim et al., 2003). Extensive studies of the PHAN expression in diverse compound-leafed species suggest that the function of PHAN in maintaining leaf adaxial identity is associated with leaflet formation in compound leaves and reduced adaxial identity of leaf primordia by down-regulation of PHAN could change pinnate compound leaves into palmate leaves (Kim et al., 2003). In pea (Pisum sativum), the role of PHAN in compound leaf development has also been elucidated by characterization of the phan mutant crispa (cri; Tattersall et al., 2005). However, unlike antisense PHAN transgenic tomato leaves, the cri mutant has the individual leaflet abaxialized, rather than the whole leaf. The number of lateral organs on the cri mutant compound leaves, including leaflets, is not altered, and the leaves remain pinnate. Apart from leaf development, the cri mutation also affects flower development. Although the floral organ identity and organ number are not altered, the laminar floral organ display abaxialized identity (Tattersall et al., 2005).The ta-siRNA pathway plays a critical role in simple-leafed species, but its role in compound-leafed species is not understood. Here, we address this question by analyzing loss-of-function reduced leaflet (rel1) and rel3 mutants in the compound-leafed species Lotus japonicus. Phenotypic characterization shows compound leaves of rel mutants exhibit a conspicuous disturbance in leaflet polarity as well as reduction in leaflet number. Besides the abnormal compound leaves, flower development is also severely affected in rel mutants, showing radial symmetric petals. REL1 and REL3 were identified by map-based cloning and were shown to be homologs of Arabidopsis SGS3 and AGO7, respectively. REL1 and REL3 act in the same genetic pathway and are both required for the biogenesis of TAS3 ta-siRNA. Further investigation reveals that the homolog of the Arabidopsis ARF3 is duplicated in the L. japonicus genome and that the duplicate ARF3 homologs and the ARF4 homolog are all negatively regulated by the ta-siRNA pathway. Furthermore, we found that the expression of LjYAB1, a homolog of Arabidopsis YAB1, was decreased in rel mutants, which may be associated with the reduced lamina.Taken together, our data reveal that the ta-siRNA pathway is integrated into the regulatory networks in the control of lateral organ development in L. japonicus and further emphasize the importance of the ta-siRNA pathway in compound leaf development. Moreover, our results also indicate many parallels between L. japonicus and monocots for the ta-siRNA pathway in the regulation of lateral organs.     

Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development

Higher plants have evolved multiple RNA-dependent RNA polymerases (RDRs), which work with Dicer-like (DCL) proteins to produce different classes of small RNAs with specialized molecular functions. Here we report that OsRDR6, the rice (Oryza sativa L.) homolog of Arabidopsis RDR6, acts in the biogenesis of various types and sizes of small RNAs. We isolated a rice osrdr6-1 mutant, which was temperature sensitive and showed spikelet defects. This mutant displays reduced accumulation of tasiR-ARFs, the conserved trans-acting siRNAs (tasiRNAs) derived from the TAS3 locus, and ectopic expression of tasiR-ARF target genes, the Auxin Response Factors (including ARF2 and ARF3/ETTIN). The loss of tasiR-mediated repression of ARFs in osrdr6-1 can explain its morphological defects, as expression of two non-targeted ARF3 gene constructs (ARF3muts) in a wild-type background mimics the osrdr6 and osdcl4-1 mutant phenotypes. Small RNA high-throughput sequencing also reveals that besides tasiRNAs, 21-nucleotide (nt) phased small RNAs are also largely dependent on OsRDR6. Unexpectedly, we found that osrdr6-1 has a strong impact on the accumulation of 24-nt phased small RNAs, but not on unphased ones. Our work uncovers the key roles of OsRDR6 in small RNA biogenesis and directly illustrates the crucial functions of tasiR-ARFs in rice development.     

Roles of DCL4 and DCL3b in rice phased small RNA biogenesis

Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5' rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice.