Argonaute Genes Research Articles

Knockdown of RNA interference pathway genes in western corn rootworm, Diabrotica virgifera virgifera, identifies no fitness costs associated with Argonaute 2 or Dicer-2

The use of transgenic crops that induce silencing of essential genes using double-stranded RNA (dsRNA) through RNA interference (RNAi) in western corn rootworm, Diabrotica virgifera virgifera, is likely to be an important component of new technologies for the control of this important corn pest. Previous studies have demonstrated that the dsRNA response in D. v. virgifera depends on the presence of RNAi pathway genes including Dicer-2 and Argonaute 2, and that downregulation of these genes limits the lethality of environmental dsRNA. A potential resistance mechanism to lethal dsRNA may involve loss of function of RNAi pathway genes. Howver, the potential for resistance to evolve may depend on whether these pathway genes have essential functions such that the loss of function of core proteins in the RNAi pathway will have fitness costs in D. v. virgifera. Fitness costs associated with potential resistance mechanisms have a central role in determining how resistance can evolve to RNAi technologies in western corn rootworm. We evaluated the effect of dsRNA and microRNA pathway gene knockdown on the development of D. v. virgifera larvae through short-term and long-term exposures to dsRNA for Dicer and Argonaute genes. Downregulation of Argonaute 2, Dicer-2, Dicer-1 did not significantly affect larval survivorship or development through short and long-term exposure to dsRNA. However, downregulation of Argonaute 1 reduced larval survivorship and delayed development. The implications of these results as they relate to D. v. virgifera resistance to lethal dsRNA are discussed.

Distinct fitness costs associated with the knockdown of RNAi pathway genes in western corn rootworm adults.

RNA interference (RNAi) based approaches can potentially be used to control insect pests. These approaches may depend on the usage of microRNA (miRNA) or double stranded RNA (dsRNA) mediated gene knockdown, which likely involves proteins that regulate these pathways, such as Argonaute 1 (Ago1), Argonaute 2 (Ago2), Dicer 1 (Dcr1), Dicer 2 (Dcr2), and Drosha in insects. We previously performed functional characterization of Ago2 and Dcr2 of western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) and observed that knockdown of Ago2 and Dcr2 ameliorated the lethal effect induced by the dsRNA-mediated knockdown of an essential gene in WCR, thereby confirming the involvement of Ago2 and Dcr2 in the dsRNA pathway. In the current study, we identified and characterized additional members of the Argonaute and Dicer gene families, namely Ago1, Ago3, Aubergine, and Dcr1, in a previously developed WCR transcriptome. We also identified a Drosha homolog in the same transcriptome. We evaluated the impacts on WCR adult fitness associated with the dsRNA-mediated knockdown of Ago1, Ago2, Dcr1, Dcr2, and Drosha genes. Among these putative RNAi pathway genes, only the knockdown of Ago1 incurred significant fitness costs such as reduced survival and oviposition rate, as well as decreased egg viability. The present study, to our knowledge, represents the first report showing that Ago1 is critical to the survival of insect adults. Our findings suggest that Ago1 plays an essential role in broader life stages of an insect than previously thought. Importantly, since fitness costs were not observed, downregulation or loss of function of RNAi pathway genes such as Ago2 or Dcr2 may confer resistance to pest control measures that rely on the normal functions of these genes. However, the precise roles of these genes under field conditions (i.e., in the presence of possible viral pathogens) requires further investigation.

Suppression of microRNA Activity in Kidney Collecting Ducts Induces Partial Loss of Epithelial Phenotype and Renal Fibrosis.

microRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. The physiologic function of these noncoding RNAs in postnatal renal tubules still remains unclear. Surprisingly, they appear to be dispensable for mammalian proximal tubule (PT) function. Here, we examined the effects of miRNA suppression in collecting ducts (CDs). To conclusively evaluate the role of miRNAs, we generated three mouse models with CD-specific inactivation of key miRNA pathway genes Dicer, Dgcr8, and the entire Argonaute gene family (Ago1, 2, 3, and 4). Characterization of these three mouse models revealed that inhibition of miRNAs in CDs spontaneously evokes a renal tubule injury-like response, which culminates in progressive tubulointerstitial fibrosis (TIF) and renal failure. Global miRNA profiling of microdissected renal tubules showed that miRNAs exhibit segmental distribution along the nephron and CDs. In particular, the expression of miR-200c is nearly 70-fold higher in CDs compared with PTs. Accordingly, miR-200s are downregulated in Dicer-KO CDs, its direct target genes Zeb1, Zeb2, and Snail2 are upregulated, and miRNA-depleted CDs undergo partial epithelial-to-mesenchymal transition (EMT). Thus, miRNAs are essential for CD homeostasis. Downregulation of CD-enriched miRNAs and the subsequent induction of partial EMT may be a new mechanism for TIF progression.

Conservation and divergence of small RNA pathways and microRNAs in land plants

BackgroundAs key regulators of gene expression in eukaryotes, small RNAs have been characterized in many seed plants, and pathways for their biogenesis, degradation, and action have been defined in model angiosperms. However, both small RNAs themselves and small RNA pathways are not well characterized in other land plants such as lycophytes and ferns, preventing a comprehensive evolutionary perspective on small RNAs in land plants.ResultsUsing 25 representatives from major lineages of lycophytes and ferns, most of which lack sequenced genomes, we characterized small RNAs and small RNA pathways in these plants. We identified homologs of DICER-LIKE (DCL), ARGONAUTE (AGO), and other genes involved in small RNA pathways, predicted over 2600 conserved microRNA (miRNA) candidates, and performed phylogenetic analyses on small RNA pathways as well as miRNAs. Pathways underlying miRNA biogenesis, degradation, and activity were established in the common ancestor of land plants, but the 24-nucleotide siRNA pathway that guides DNA methylation is incomplete in sister species of seed plants, especially lycophytes. We show that the functional diversification of key gene families such as DCL and AGO as observed in angiosperms occurred early in land plants followed by parallel expansion of the AGO family in ferns and angiosperms. We uncovered a conserved AGO subfamily absent in angiosperms.ConclusionsOur phylogenetic analyses of miRNAs in bryophytes, lycophytes, ferns, and angiosperms refine the time-of-origin for conserved miRNA families as well as small RNA machinery in land plants.

Differential expression of small RNA pathway genes associated with the Biomphalaria glabrata/Schistosoma mansoni interaction.

The World Health Organization (WHO) estimates that approximately 240 million people in 78 countries require treatment for schistosomiasis, an endemic disease caused by trematodes of the genus Schistosoma. In Brazil, Schistosoma mansoni is the only species representative of the genus whose passage through an invertebrate host, snails of the genus Biomphalaria, is obligatory before infecting a mammalian host, including humans. The availability of the genome and transcriptome of B. glabrata makes studying the regulation of gene expression, particularly the regulation of miRNA and piRNA processing pathway genes, possible. This might assist in better understanding the biology of B. glabrata as well as its relationship to the parasite S. mansoni. Some aspects of this interaction are still poorly explored, including the participation of non-coding small RNAs, such as miRNAs and piRNAs, with lengths varying from 18 to 30 nucleotides in mature form, which are potent regulators of gene expression. Using bioinformatics tools and quantitative PCR, we characterized and validated the miRNA and piRNA processing pathway genes in B. glabrata. In silico analyses showed that genes involved in miRNA and piRNA pathways were highly conserved in protein domain distribution, catalytic site residue conservation and phylogenetic analysis. Our study showed differential expression of putative Argonaute, Drosha, Piwi, Exportin-5 and Tudor genes at different snail developmental stages and during infection with S. mansoni, suggesting that the machinery is required for miRNA and piRNA processing in B. glabrata at all stages. These data suggested that the silencing pathway mediated by miRNAs and piRNAs can interfere in snail biology throughout the life cycle of the snail, thereby influencing the B. glabrata/S. mansoni interaction. Further studies are needed to confirm the participation of the small RNA processing pathway proteins in the parasite/host relationship, mainly the effective participation of small RNAs in regulating their target genes.

The Conserved RNA Binding Cyclophilin, Rct1, Regulates Small RNA Biogenesis and Splicing Independent of Heterochromatin Assembly

RNAi factors and their catalytic activities are essential for heterochromatin assembly in S.pombe. This has led to the idea that siRNAs can promote H3K9 methylation by recruiting the cryptic loci regulator complex (CLRC), also known as recombination inKcomplex (RIKC), to the nucleation site. The conserved RNA-binding protein Rct1 (AtCyp59/SIG-7) interacts with splicing factors and RNA polymerase II. Here we show that Rct1 promotes processing of pericentromeric transcripts into siRNAs via the RNA recognition motif. Surprisingly, loss of siRNA in rct1 mutants has no effect on H3K9 di- or tri-methylation, resembling other splicing mutants, suggesting that post-transcriptional gene silencing per se is not required to maintain heterochromatin. Splicing of the Argonaute gene is also defective in rct1 mutants and contributes to loss of silencing but not to loss of siRNA. Our results suggest thatRct1 guides transcripts to the RNAi machinery by promoting splicing of elongating non-coding transcripts.

Expression analysis of argonaute, Dicer-like, and RNA-dependent RNA polymerase genes in cucumber (Cucumis sativus L.) in response to abiotic stress.

Posttranscriptional control of gene expression can be achieved through RNA interference when the activities of Dicer-like (DCL), argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins are significant. In this study, we analysed the expression of seven AGO, five DCL and eight RDR genes in cucumber under cold, heat, hormone, salinity and dehydration treatments using quantitative reverse-transcription PCR (qRT-PCR). All CsAGO, CsDCL and CsRDR genes were differentially expressed under abiotic stress treatment. In response to abiotic stress treatment, most genes were expressed at higher levels in flowers or stems than in other organs, whereas some CsAGOs (CsAGO1c, CsAGO6 and CsAGO7) and CsRDRs (CsRDR1d and CsRDR2) were highly expressed in roots during dehydration treatment. The expression patterns indicate that most CsDCLs, CsAGOs and CsRDRs respond to abiotic stress, and stems or flowers are the most sensitive organs, followed by roots. This is the first report of expression analysis of all CsDCL, CsAGO and CsRDR family genes in cucumber under abiotic stress, which provides basic information and insights into the putative roles of these genes in abiotic stress. The results of this study should serve as a basis for further functional characterization of these gene families in cucumber and related Cucurbitaceae species.

SRNAs as possible regulators of retrotransposon activity in Cryptococcus gattii VGII

BackgroundThe absence of Argonaute genes in the fungal pathogen Cryptococcus gattii R265 and other VGII strains indicates that yeasts of this genotype cannot have a functional RNAi pathway, an evolutionarily conserved gene silencing mechanism performed by small RNAs. The success of the R265 strain as a pathogen that caused the Pacific Northwest and Vancouver Island outbreaks may imply that RNAi machinery loss could be beneficial under certain circumstances during evolution. As a result, a hypermutant phenotype would be created with high rates of genome retrotransposition, for instance. This study therefore aimed to evaluate in silicio the effect of retrotransposons and their control mechanisms by small RNAs on genomic stability and synteny loss of C. gattii R265 through retrotransposons sequence comparison and orthology analysis with other 16 C. gattii genomic sequences available.ResultsRetrotransposon mining identified a higher sequence count to VGI genotype compared to VGII, VGIII, and VGIV. However, despite the lower retrotransposon number, VGII exhibited increased synteny loss and genome rearrangement events. RNA-Seq analysis indicated highly expressed retrotransposons as well as sRNA production.ConclusionsGenome rearrangement and synteny loss may suggest a greater retrotransposon mobilization caused by RNAi pathway absence, but the effective presence of sRNAs that matches retrotransposon sequences means that an alternative retrotransposon silencing mechanism could be active in genomic integrity maintenance of C. gattii VGII strains.

Dicer and Argonaute Genes Involved in RNA Interference in the Entomopathogenic Fungus Metarhizium robertsii.

RNA interference (RNAi) is a gene-silencing mechanism that plays an important role in gene regulation in a number of eukaryotic organisms. Two core components, Dicer and Argonaute, are central in the RNAi machinery. However, the physiological roles of Dicer and Argonaute in the entomopathogenic fungus Metarhizium robertsii have remained unclear. Here, the roles of genes encoding Dicer (M. robertsiidcl1 [Mrdcl1] and Mrdcl2) and Argonaute (Mrago1 and Mrago2) proteins in M. robertsii were investigated. The results showed that the Dicer-like protein MrDCL2 and Argonaute protein MrAGO1 are the major components of the RNAi process occurring in M. robertsii The Dicer and Argonaute genes were not involved in the regulation of growth and diverse abiotic stress response in M. robertsii under the tested conditions. Moreover, our results showed that the Dicer and Argonaute gene mutants demonstrated reduced abilities to produce conidia, compared to the wild type (WT) and the gene-rescued mutant. In particular, the conidial yields in the Δdcl2 and Δago1 mutants were reduced by 55.8% and 59.3%, respectively, compared with those from the control strains. Subsequently, for the WT and Δdcl2 mutant strains, digital gene expression (DGE) profiling analysis of the stage of mycelium growth and conidiogenesis revealed that modest changes occur in development or metabolism processes, which may explain the reduction in conidiation in the Δdcl2 mutant. In addition, we further applied high-throughput sequencing technology to identify small RNAs (sRNAs) that are differentially expressed in the WT and the Δdcl2 mutant and found that 4 known microRNA-like small RNAs (milRNAs) and 8 novel milRNAs were Mrdcl2 dependent in M. robertsiiIMPORTANCE The identification and characterization of components in RNAi have contributed significantly to our understanding of the mechanism and functions of RNAi in eukaryotes. Here, we found that Dicer and Argonaute genes play an important role in regulating conidiation in M. robertsii Our study also demonstrates that diverse small RNA pathways exist in M. robertsii The study provides a theoretical platform for exploration of the functions of Dicer and Argonaute genes involved in RNAi in fungi.

Abscisic Acid Induces Resistance against Bamboo Mosaic Virus through Argonaute2 and 3.

Plant resistance to pathogens is tuned by defense-related hormones. Of these, abscisic acid (ABA) is well documented to moderate resistance against fungi and bacteria. However, ABA's contribution to resistance against viruses is pleiotropic. ABA affects callose deposition at plasmodesmata (therefore hindering the viral cell-to-cell movement), but here, we show that when callose synthase is down-regulated, ABA still induces resistance against infection with Bamboo mosaic virus (BaMV). By examining the potential connections between the ABA and RNA-silencing pathways in Arabidopsis (Arabidopsis thaliana), we showed that ABA regulates the expression of almost the whole ARGONAUTE (AGO) gene family, of which some are required for plant resistance against BaMV Our data show that BaMV infection and ABA treatment regulate the same set of AGOs, with positive effects on AGO1, AGO2, and AGO3, no effect on AGO7, and negative effects on AGO4 and AGO10 The BaMV-mediated regulation of AGO1, AGO2, and AGO3 is ABA dependent, because the accumulation of these AGOs in BaMV-infected ABA mutants did not reach the levels observed in infected wild-type plants. In addition, the AGO1-miR168a complex is dispensable for BaMV resistance, while AGO2 and AGO3 were important for ABA-mediated resistance. While most ago mutants showed increased susceptibility to BaMV infection (except ago10), ago1-27 showed reduced BaMV titers, which was attributed to the up-regulated levels of AGO2, AGO3, and AGO4 We have established that ABA regulates the expression of several members of the AGO family, and this regulation partially contributes to ABA-mediated resistance against BaMV These findings reveal another role for ABA in plants.

The Evolutionary Loss of RNAi Key Determinants in Kinetoplastids as a Multiple Sporadic Phenomenon.

We screened the genomes of a broad panel of kinetoplastid protists for genes encoding proteins associated with the RNA interference (RNAi) system using probes from the Argonaute (AGO1), Dicer1 (DCL1), and Dicer2 (DCL2) genes of Leishmania brasiliensis and Crithidia fasciculata. We identified homologs for all the three of these genes in the genomes of a subset of these organisms. However, several of these organisms lacked evidence for any of these genes, while others lacked only DCL2. The open reading frames encoding these putative proteins were structurally analyzed in silico. The alignments indicated that the genes are homologous with a high degree of confidence, and three-dimensional structural models strongly supported a functional relationship to previously characterized AGO1, DCL1, and DCL2 proteins. Phylogenetic analysis of these putative proteins showed that these genes, when present, evolved in parallel with other nuclear genes, arguing that the RNAi system genes share a common progenitor, likely across all Kinetoplastea. In addition, the genome segments bearing these genes are highly conserved and syntenic, even among those taxa in which they are absent. However, taxa in which these genes are apparently absent represent several widely divergent branches of kinetoplastids, arguing that these genes were independently lost at least six times in the evolutionary history of these organisms. The mechanisms responsible for the apparent coordinate loss of these RNAi system genes independently in several lineages of kinetoplastids, while being maintained in other related lineages, are currently unknown.

Cloning and Characterization of Argonaute Genes in Tomato.

Argonaute (AGO) proteins are core elements in plant posttranscriptional RNA silencing pathways. The identification and functional characterization of tomato (Solanum lycopersicum) AGOs will help to better understand RNA silencing-based posttranscriptional regulation in fleshy fruits. Here we describe how to identify and clone SlAGO genes, as well as the methodology for their functional characterization.

The LBD12-1 Transcription Factor Suppresses Apical Meristem Size by Repressing Argonaute 10 Expression.

The shoot apical meristem (SAM) consists of a population of multipotent cells that generates all aerial structures and regenerates itself. SAM maintenance and lateral organ development are regulated by several complex signaling pathways, in which the Argonaute gene-mediated pathway plays a key role. One Argonaute gene, AGO10, functions as a microRNA locker that attenuates miR165/166 activity and positively regulates shoot apical meristem development, but little is known about when and how AGO10 is regulated at the transcriptional level. In this work, we showed that transgenic rice plants overexpressing LBD12-1, an LBD family transcription factor, exhibited stunted growth, twisted leaves, abnormal anthers, and reduced SAM size. Further research revealed that LBD12-1 directly binds to the promoter region and represses the expression of AGO10. Overexpression of AGO10 in an LBD12-1 overexpression background rescued the growth defect phenotype of LBD12-1-overexpressing plants. The expression of LBD12-1 and its binding ability to the AGO10 promoter is induced by stress. lbd12-1 loss-of-function mutants showed similar phenotypes and SAM size to the wild type under normal conditions, but lbd12-1 had a larger SAM under salt stress. Our findings provide novel insights into the regulatory mechanism of AGO10 by which SAM size is controlled under stress conditions.

A Multigenic Network of ARGONAUTE4 Clade Members Controls Early Megaspore Formation in Arabidopsis

The development of gametophytes relies on the establishment of a haploid gametophytic generation that initiates with the specification of gametophytic precursors. The majority of flowering plants differentiate a single gametophytic precursor in the ovule: the megaspore mother cell. Here we show that, in addition to argonaute9 (ago9), mutations in other ARGONAUTE (AGO) genes such as ago4, ago6, and ago8, also show abnormal configurations containing supernumerary gametophytic precursors in Arabidopsis thaliana. Double homozygous ago4 ago9 individuals showed a suppressive effect on the frequency of ovules with multiple gametophytic precursors across three consecutive generations, indicating that genetic interactions result in compensatory mechanisms. Whereas overexpression of AGO6 in ago9 and ago4 ago9 confirms strong regulatory interactions among genes involved in RNA-directed DNA methylation, AGO8 is overexpressed in premeiotic ovules of ago4 ago9 individuals, suggesting that the regulation of this previously presumed pseudogene responds to the compensatory mechanism. The frequency of abnormal meiotic configurations found in ago4 ago9 individuals is dependent on their parental genotype, revealing a transgenerational effect. Our results indicate that members of the AGO4 clade cooperatively participate in preventing the abnormal specification of multiple premeiotic gametophytic precursors during early ovule development in A. thaliana.

Genome-wide identification of Argonaute 1- and Argonaute 2-regulating genes revealed an inhibition of macula-like virus by RNAi pathway in the silkworm, Bombyx mori

Genome-wide identification of Argonaute 1- and Argonaute 2-regulating genes revealed an inhibition of macula-like virus by RNAi pathway in the silkworm, Bombyx mori

Generation of an integrated Hieracium genomic and transcriptomic resource enables exploration of small RNA pathways during apomixis initiation.

BackgroundApplication of apomixis, or asexual seed formation, in crop breeding would allow rapid fixation of complex traits, economizing improved crop delivery. Identification of apomixis genes is confounded by the polyploid nature, high genome complexity and lack of genomic sequence integration with reproductive tissue transcriptomes in most apomicts.ResultsA genomic and transcriptomic resource was developed for Hieracium subgenus Pilosella (Asteraceae) which incorporates characterized sexual, apomictic and mutant apomict plants exhibiting reversion to sexual reproduction. Apomicts develop additional female gametogenic cells that suppress the sexual pathway in ovules. Disrupting small RNA pathways in sexual Arabidopsis also induces extra female gametogenic cells; therefore, the resource was used to examine if changes in small RNA pathways correlate with apomixis initiation. An initial characterization of small RNA pathway genes within Hieracium was undertaken, and ovary-expressed ARGONAUTE genes were identified and cloned. Comparisons of whole ovary transcriptomes from mutant apomicts, relative to the parental apomict, revealed that differentially expressed genes were enriched for processes involved in small RNA biogenesis and chromatin silencing. Small RNA profiles within mutant ovaries did not reveal large-scale alterations in composition or length distributions; however, a small number of differentially expressed, putative small RNA targets were identified.ConclusionsThe established Hieracium resource represents a substantial contribution towards the investigation of early sexual and apomictic female gamete development, and the generation of new candidate genes and markers. Observed changes in small RNA targets and biogenesis pathways within sexual and apomictic ovaries will underlie future functional research into apomixis initiation in Hieracium.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0311-0) contains supplementary material, which is available to authorized users.

Comprehensive genomic analysis and expression profiling of Argonaute gene family and examination of their regulatory roles in water-use efficiency and abiotic stress responses in apple

Argonaute proteins are key players in small RNA-guided gene-silencing, which plays an important role in plant stress tolerance. However, little is known about how Argonaute genes affect the water-use efficiency (WUE) of apple (Malus domestica) or its responses to different abiotic stresses. We identified and characterized MdAGOs in apple and analyzed their chromosome locations, exon/intron structures, phylogeny, and the distribution of conserved motifs. We also examined the expression profiling of responses to abiotic stress and conducted molecular cloning of MdAGO4.1. In all, 16 MdAGOs were identified and characterized, then grouped into three separate clusters. Our qRT-PCR data demonstrated that these genes are induced by drought, salt, cold, and ABA treatments, indicating that they are good candidates for further analysis of their activities and functions. We have previously shown that, during long-term moderate drought, the abundance of MdAGO4.1 protein is increased in ‘Qinguan’ apple (a cultivar with high WUE) leaves, but not in ‘Naganofuji No. 2’. These changes are in accordance with alterations in their WUE and expression of MdAGO4.1 under the same test conditions as those used in the current study. Therefore, MdAGO4.1 is a putative gene that positively regulates WUE. Our findings provide evidence that MdAGOs have a role in plant adaptations to abiotic stress and can be exploited to improve WUE. These results will serve as a framework for future functional studies of that gene family in apple.

Genomic identification, rapid evolution, and expression of Argonaute genes in the tilapia, Oreochromis niloticus.

Argonaute proteins are key components of the small RNA-induced silencing complex and have multiple roles in RNA-directed regulatory pathways. Argonaute genes can be divided into two subfamilies: the Ago (interacting with microRNA/small interfering RNA) and Piwi subfamilies (interacting with piwi-interacting RNAs (piRNAs)). In the present study, genome-wide analyses firstly yielded the identification of different members of Agos and Piwis in the tilapia, coelacanth, spotted gar, and elephant shark. The additional teleost Ago3b was generated following the fish-specific genome duplication event. Selective pressure analysis on Agos and Piwis between cichlids and other teleosts showed an accelerated evolution of Piwil1 in the cichlid lineages, and the positive selected sites were located in the region of PIWI domain, suggesting that these amino acid substitutions are adapt to targeted cleavage of messenger RNA (mRNA) in cichlids. Ago1 and Ago4 were detected at higher levels at 5days after hatching (dah) in both ovaries and testes compared with other stages, supporting the previously reported requirement of Ago-mediated pathways to clear the maternal mRNAs during the early embryogenesis. The Piwis were abundantly expressed in tilapia testes, indicating their essential roles in male germline, especially in spermatogenesis. Notable expression of Piwis was also detected in skeletal muscle, indicating that piRNA pathway may not only be confined to development and maintenance of the germline but may also play important roles in somatic tissues. The expression of Piwil1 and Piwil2 was examined by quantitative PCR (qPCR) and in situ hybridization (ISH) to validate the spatial and temporal expression profiles. Taken together, these results present a thorough overview of tilapia Argonaute family and provide a new perspective on the evolution and function of this family in teleosts.

Identification and functional characterization of the AGO1 ortholog in maize.

Eukaryotic Argonaute proteins play primary roles in miRNA and siRNA pathways that are essential for numerous developmental and biological processes. However, the functional roles of the four ZmAGO1 genes have not yet been characterized in maize (Zea mays L.). In the present study, ZmAGO1a was identified from four putative ZmAGO1 genes for further characterization. Complementation of the Arabidopsis ago1‐27 mutant with ZmAGO1a indicated that constitutive overexpression of ZmAGO1a could restore the smaller rosette, serrated leaves, later flowering and maturation, lower seed set, and darker green leaves at late stages of the mutant to the wild‐type phenotype. The expression profiles of ZmAGO1a under five different abiotic stresses indicated that ZmAGO1a shares expression patterns similar to those of Argonaute genes in rice, Arabidopsis, and wheat. Further, variation in ZmAGO1a alleles among diverse maize germplasm that resulted in several amino acid changes revealed genetic diversity at this locus. The present data suggest that ZmAGO1a might be an important AGO1 ortholog in maize. The results presented provide further insight into the function of ZmAGO1a.

Duplication and Diversification of Dipteran Argonaute Genes, and the Evolutionary Divergence of Piwi and Aubergine.

Genetic studies of Drosophila melanogaster have provided a paradigm for RNA interference (RNAi) in arthropods, in which the microRNA and antiviral pathways are each mediated by a single Argonaute (Ago1 and Ago2) and germline suppression of transposable elements is mediated by a trio of Piwi-subfamily Argonaute proteins (Ago3, Aub, and Piwi). Without a suitable evolutionary context, deviations from this can be interpreted as derived or idiosyncratic. Here we analyze the evolution of Argonaute genes across the genomes and transcriptomes of 86 Dipteran species, showing that variation in copy number can occur rapidly, and that there is constant flux in some RNAi mechanisms. The lability of the RNAi pathways is illustrated by the divergence of Aub and Piwi (182–156 Ma), independent origins of multiple Piwi-family genes in Aedes mosquitoes (less than 25Ma), and the recent duplications of Ago2 and Ago3 in the tsetse fly Glossina morsitans. In each case the tissue specificity of these genes has altered, suggesting functional divergence or innovation, and consistent with the action of dynamic selection pressures across the Argonaute gene family. We find there are large differences in evolutionary rates and gene turnover between pathways, and that paralogs of Ago2, Ago3, and Piwi/Aub show contrasting rates of evolution after duplication. This suggests that Argonautes undergo frequent evolutionary expansions that facilitate functional divergence.