Xianwei Song

Degradome sequencing reveals endogenous small RNA targets in rice (Oryza sativa L. ssp. indica)

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) regulate gene expression in eukaryotes. Plant miRNAs modulate their targets mainly via messenger RNA (mRNA) cleavage. Small RNA (sRNA) targets have been extensively investigated in Arabidopsis using computational prediction, experimental validation, and degradome sequencing. However, small RNA targets are largely unknown in rice (Oryza sativa). Here, we report global identification of small RNA targets using high throughput degradome sequencing in the rice indica cultivar 93–11 (Oryza sativa L. ssp. indica). One hundred and seventy-seven transcripts targeted by a total of 87 unique miRNAs were identified. Of targets for the conserved miRNAs between Arabidopsis and rice, transcription factors comprise around 70% (58 in 82), indicating that these miRNAs act as masters of gene regulatory nodes in rice. In contrast, non-conserved miRNAs targeted diverse genes which provide more complex regulatory networks. In addition, 5 AUXIN RESPONSE FACTORs (ARFs) cleaved by the TAS3 derived ta-siRNAs were also detected. A total of 40 sRNA targets were further validated via RNA ligasemediated 5’ rapid amplification of cDNA ends (RLM 5’-RACE). Our degradome results present a detailed sRNA-target interaction atlas, which provides a guide for the study of the roles of sRNAs and their targets in rice.

Oryza sativa Dicer-like4 Reveals a Key Role for Small Interfering RNA Silencing in Plant Development

MicroRNAs and small interfering RNAs (siRNAs) are two classes of small regulatory RNAs derived from different types of precursors and processed by distinct Dicer or Dicer-like (DCL) proteins. During evolution, four Arabidopsis thaliana DCLs and six rice (Oryza sativa) DCLs (Os DCLs) appear to have acquired specialized functions. The Arabidopsis DCLs are well characterized, but those in rice remain largely unstudied. Here, we show that both knockdown and loss of function of rice DCL4, the homolog of Arabidopsis DCL4, lead to vegetative growth abnormalities and severe developmental defects in spikelet identity. These phenotypic alterations appear to be distinct from those observed in Arabidopsis dcl4 mutants, which exhibit accelerated vegetative phase change. The difference in phenotype between rice and Arabidopsis dcl4 mutants suggests that siRNA processing by DCL4 has a broader role in rice development than in Arabidopsis. Biochemical and genetic analyses indicate that Os DCL4 is the major Dicer responsible for the 21-nucleotide siRNAs associated with inverted repeat transgenes and for trans-acting siRNA (ta-siRNA) from the endogenous TRANS-ACTING siRNA3 (TAS3) gene. We show that the biogenesis mechanism of TAS3 ta-siRNA is conserved but that putative direct targets of Os DCL4 appear to be differentially regulated between monocots and dicots. Our results reveal a critical role of Os DCL4-mediated ta-siRNA biogenesis in rice development.

NOT2 Proteins Promote Polymerase II–Dependent Transcription and Interact with Multiple MicroRNA Biogenesis Factors in Arabidopsis

This work identifies a pair of evolutionarily conserved NOT2_3_5 domain–containing proteins that act as a scaffold to interact with polymerase II and some pri-miRNA processing proteins, thus promoting the transcription of protein-coding and MIR genes and facilitating efficient DCL1 recruitment in miRNA biogenesis. MicroRNAs (miRNAs) play key regulatory roles in numerous developmental and physiological processes in animals and plants. The elaborate mechanism of miRNA biogenesis involves transcription and multiple processing steps. Here, we report the identification of a pair of evolutionarily conserved NOT2_3_5 domain–containing-proteins, NOT2a and NOT2b (previously known as At-Negative on TATA less2 [NOT2] and VIRE2-INTERACTING PROTEIN2, respectively), as components involved in Arabidopsis thaliana miRNA biogenesis. NOT2 was identified by its interaction with the Piwi/Ago/Zwille domain of DICER-LIKE1 (DCL1), an interaction that is conserved between rice (Oryza sativa) and Arabidopsis thaliana. Inactivation of both NOT2 genes in Arabidopsis caused severe defects in male gametophytes, and weak lines show pleiotropic defects reminiscent of miRNA pathway mutants. Impairment of NOT2s decreases the accumulation of primary miRNAs and mature miRNAs and affects DCL1 but not HYPONASTIC LEAVES1 (HYL1) localization in vivo. In addition, NOT2b protein interacts with polymerase II and other miRNA processing factors, including two cap binding proteins, CBP80/ABH1, CBP20, and SERRATE (SE). Finally, we found that the mRNA levels of some protein coding genes were also affected. Therefore, these results suggest that NOT2 proteins act as general factors to promote the transcription of protein coding as well as miRNA genes and facilitate efficient DCL1 recruitment in miRNA biogenesis.

Dicer-like 3 produces transposable element-associated 24-nt siRNAs that control agricultural traits in rice

Significance The functional relationship of transposons and small RNAs remains an important question in the study of gene expression and its effect on agronomic traits. Here, we use deep sequencing of small RNAs to provide the first evidence that the rice Dicer-like 3 homolog OsDCL3a produces 24-nt small interfering RNAs (siRNAs) predominantly associated with miniature inverted repeat transposable elements (MITEs). These 24-nt siRNAs target genes adjacent to MITEs and act as broadly functioning regulators of gene expression. In particular, OsDCL3a directly targets genes involved in homeostasis of the plant hormones gibberellin and brassinosteroid, thus controlling important agricultural traits. This mechanism of fine-tuning gene expression mediated by MITEs may be conserved in organisms with genomes rich in dispersed repeats or transposable elements. Transposable elements (TEs) and repetitive sequences make up over 35% of the rice (Oryza sativa) genome. The host regulates the activity of different TEs by different epigenetic mechanisms, including DNA methylation, histone H3K9 methylation, and histone H3K4 demethylation. TEs can also affect the expression of host genes. For example, miniature inverted repeat TEs (MITEs), dispersed high copy-number DNA TEs, can influence the expression of nearby genes. In plants, 24-nt small interfering RNAs (siRNAs) are mainly derived from repeats and TEs. However, the extent to which TEs, particularly MITEs associated with 24-nt siRNAs, affect gene expression remains elusive. Here, we show that the rice Dicer-like 3 homolog OsDCL3a is primarily responsible for 24-nt siRNA processing. Impairing OsDCL3a expression by RNA interference caused phenotypes affecting important agricultural traits; these phenotypes include dwarfism, larger flag leaf angle, and fewer secondary branches. We used small RNA deep sequencing to identify 535,054 24-nt siRNA clusters. Of these clusters, ∼82% were OsDCL3a-dependent and showed significant enrichment of MITEs. Reduction of OsDCL3a function reduced the 24-nt siRNAs predominantly from MITEs and elevated expression of nearby genes. OsDCL3a directly targets genes involved in gibberellin and brassinosteroid homeostasis; OsDCL3a deficiency may affect these genes, thus causing the phenotypes of dwarfism and enlarged flag leaf angle. Our work identifies OsDCL3a-dependent 24-nt siRNAs derived from MITEs as broadly functioning regulators for fine-tuning gene expression, which may reflect a conserved epigenetic mechanism in higher plants with genomes rich in dispersed repeats or TEs.

Control of transposon activity by a histone H3K4 demethylase in rice

Transposable elements (TEs) are ubiquitously present in plant genomes and often account for significant fractions of the nuclear DNA. For example, roughly 40% of the rice genome consists of TEs, many of which are retrotransposons, including 14% LTR- and ∼1% non-LTR retrotransposons. Despite their wide distribution and abundance, very few TEs have been found to be transpositional, indicating that TE activities may be tightly controlled by the host genome to minimize the potentially mutagenic effects associated with active transposition. Consistent with this notion, a growing body of evidence suggests that epigenetic silencing pathways such as DNA methylation, RNA interference, and H3K9me2 function collectively to repress TE activity at the transcriptional and posttranscriptional levels. It is not yet clear, however, whether the removal of histone modifications associated with active transcription is also involved in TE silencing. Here, we show that the rice protein JMJ703 is an active H3K4-specific demethylase required for TEs silencing. Impaired JMJ703 activity led to elevated levels of H3K4me3, the misregulation of numerous endogenous genes, and the transpositional reactivation of two families of non-LTR retrotransposons. Interestingly, loss of JMJ703 did not affect TEs (such as Tos17) previously found to be silenced by other epigenetic pathways. These results indicate that the removal of active histone modifications is involved in TE silencing and that different subsets of TEs may be regulated by distinct epigenetic pathways.

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.

Epigenetic Mutation of RAV6 Affects Leaf Angle and Seed Size in Rice

Epigenetic modification of a B3 transcription factor alters rice leaf angle via modulation of brassinosteroid homeostasis. Heritable epigenetic variants of genes, termed epialleles, can broaden genetic and phenotypic diversity in eukaryotes. Epialleles may also provide a new source of beneficial traits for crop breeding, but very few epialleles related to agricultural traits have been identified in crops. Here, we identified Epi-rav6, a gain-of-function epiallele of rice (Oryza sativa) RELATED TO ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1 (VP1) 6 (RAV6), which encodes a B3 DNA-binding domain-containing protein. The Epi-rav6 plants show larger lamina inclination and smaller grain size; these agronomically important phenotypes are inherited in a semidominant manner. We did not find nucleotide sequence variation of RAV6. Instead, we found hypomethylation in the promoter region of RAV6, which caused ectopic expression of RAV6 in Epi-rav6 plants. Bisulfite analysis revealed that cytosine methylation of four CG and two CNG loci within a continuous 96-bp region plays essential roles in regulating RAV6 expression; this region contains a conserved miniature inverted repeat transposable element transposon insertion in cultivated rice genomes. Overexpression of RAV6 in the wild type phenocopied the Epi-rav6 phenotype. The brassinosteroid (BR) receptor BR INSENSITIVE1 and BR biosynthetic genes EBISU DWARF, DWARF11, and BR-DEFICIENT DWARF1 were ectopically expressed in Epi-rav6 plants. Also, treatment with a BR biosynthesis inhibitor restored the leaf angle defects of Epi-rav6 plants. This indicates that RAV6 affects rice leaf angle by modulating BR homeostasis and demonstrates an essential regulatory role of epigenetic modification on a key gene controlling important agricultural traits. Thus, our work identifies a unique rice epiallele, which may represent a common phenomenon in complex crop genomes.

ROS accumulation and antiviral defence control by microRNA528 in rice.

MicroRNAs (miRNAs) are key regulators of plant–pathogen interactions. Modulating miRNA function has emerged as a new strategy to produce virus resistance traits1–5. However, the miRNAs involved in antiviral defence and the underlying mechanisms remain largely elusive. We previously demonstrated that sequestration by Argonaute (AGO) proteins plays an important role in regulating miRNA function in antiviral defence pathways6. Here we reveal that cleavage-defective AGO18 complexes sequester microRNA528 (miR528) upon viral infection. We show that miR528 negatively regulates viral resistance in rice by cleaving L-ascorbate oxidase (AO) messenger RNA, thereby reducing AO-mediated accumulation of reactive oxygen species. Upon viral infection, miR528 becomes preferentially associated with AGO18, leading to elevated AO activity, higher basal reactive oxygen species accumulation and enhanced antiviral defence. Our findings reveal a mechanism in which antiviral defence is boosted through suppression of an miRNA that negatively regulates viral resistance. This mechanism could be manipulated to engineer virus-resistant crop plants.

Integrated analysis of phenome, genome, and transcriptome of hybrid rice uncovered multiple heterosis-related loci for yield increase.

Significance Because of its practical importance and scientific significance, heterosis (hybrid vigor) is an interesting topic for both breeders and biologists. However, although heterosis has been applied successfully to increase crop yields, the molecular mechanisms involved remain obscure. In this study, using an integrative approach, we found that multiple quantitative trait loci (QTLs) cumulatively drive yield heterosis in hybrid rice by regulating two grain-yield component traits in which the RH8 (rice heterosis 8) gene plays a major role. Our research highlights the importance of integrative methods to uncover the molecular mechanism of heterosis and thus pave a way toward revealing the molecular mechanisms in rice heterosis in detail. Hybrid rice is the dominant form of rice planted in China, and its use has extended worldwide since the 1970s. It offers great yield advantages and has contributed greatly to the world’s food security. However, the molecular mechanisms underlying heterosis have remained a mystery. In this study we integrated genetics and omics analyses to determine the candidate genes for yield heterosis in a model two-line rice hybrid system, Liang-you-pei 9 (LYP9) and its parents. Phenomics study revealed that the better parent heterosis (BPH) of yield in hybrid is not ascribed to BPH of all the yield components but is specific to the BPH of spikelet number per panicle (SPP) and paternal parent heterosis (PPH) of effective panicle number (EPN). Genetic analyses then identified multiple quantitative trait loci (QTLs) for these two components. Moreover, a number of differentially expressed genes and alleles in the hybrid were mapped by transcriptome profiling to the QTL regions as possible candidate genes. In parallel, a major QTL for yield heterosis, rice heterosis 8 (RH8), was found to be the DTH8/Ghd8/LHD1 gene. Based on the shared allelic heterozygosity of RH8 in many hybrid rice cultivars, a common mechanism for yield heterosis in the present commercial hybrid rice is proposed.

An Epiallele of Rice AK1 Affects Photosynthetic Capacity

Epigenetic gene variants, termed epialleles, can broaden genetic and phenotypic diversity in eukaryotes. Here, we identify a natural epiallele of OsAK1, which encodes a rice adenylate kinase. The Epi-ak1 plants show albino in young leaf and panicle with abnormal chloroplast structures. We found that no nucleotide sequence variation but hypermethylation at promoter region caused silencing of OsAK1 (Os08g01770) in Epi-ak1 plants. OsAK1 localizes to chloroplast and many genes associated with photosynthesis processes were downregulated in Epi-ak1. Thus, the work identified a novel rice epiallele caused by DNA methylation changes, shedding light on significant roles of DNA methylation on rice development.