RNA-dependent RNA Polymerase Gene Families Research Articles

Genome-wide identification and expression analyses of the Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families in onion (Allium cepa L.)

ABSTRACT RNA silencing is a highly conserved gene expression regulatory mechanism participating in plant development and stress responses. Dicer-like protein (DCL), Argonaute protein (AGO) and RNA-dependent RNA polymerase (RDR) are directly involved in RNA silencing. So far, the characterisation and expression analysis of RDR, AGO and DCL proteins have not been performed in onion. A genome-wide analysis identified eight AcAGO, four AcDCL and four AcRDR putative genes in onion. Their cis-acting elements were investigated. The transcript levels of genes in different onion tissues and response to abiotic stress were studied. The results demonstrated that AcAGOs, AcDCLs and AcRDRs showed various tissue expression patterns. AcAGO2 and AcAGO4b were highly expressed in onion bulbs. AcDCLs and AcRDRs showed the highest transcript levels in inflorescence. AcAGO1, AcAGO5 and AcDCL1 were constitutively expressed in leaf and bulb during onion bulb expansion. AcRDR1 and AcRDR6 were upregulated during bulb expansion. AcAGOs were upregulated with salinity treatment, whereas AcDCLs and AcRDRs showed different expression patterns in abiotic stress response. Our results provided a basis for further clarification of RNA silencing-mediated environment stimulation response and development in onion.

Comprehensive analysis of Dicer-like, Argonaute, and RNA-dependent RNA polymerase gene families and their expression analysis in response to abiotic stresses in Jatropha curcas

ABSTRACT Eukaryotic gene expression is regulated by RNA interference (RNAi). Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) are essential elements involved in the process of RNAi. However, Jatropha (J.) curcas, an oil-producing plant, has not undergone any previous systematic analysis or expression profiling of these genes. In this study, a total of four JcDCL, nine JcAGO, and seven JcRDR genes were identified in the J. curcas genome. Based on phylogenetic analysis, each gene family was classified into four clades and randomly distributed on nine J. curcas chromosomes. Protein sequence alignment revealed that the classical PAZ, Piwi, and RdRP domains were present in all JcDCLs, JcAGOs, and JcRDRs, respectively. Comprehensive expression pattern analysis using RNA-seq data showed various expression patterns in different organs and under different abiotic stresses. In addition, special attention should be paid to JcDCL1, JcAGO1, JcAGO4, and JcRDR5, which play important roles in response to various stresses.

Genome-wide identification of DCL, AGO and RDR gene families and their associated functional regulatory element analyses in sunflower (Helianthus annuus).

RNA interference (RNAi) regulates a variety of eukaryotic gene expressions that are engaged in response to stress, growth, and the conservation of genomic stability during developmental phases. It is also intimately connected to the post-transcriptional gene silencing (PTGS) process and chromatin modification levels. The entire process of RNA interference (RNAi) pathway gene families mediates RNA silencing. The main factors of RNA silencing are the Dicer-Like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) gene families. To the best of our knowledge, genome-wide identification of RNAi gene families like DCL, AGO, and RDR in sunflower (Helianthus annuus) has not yet been studied despite being discovered in some species. So, the goal of this study is to find the RNAi gene families like DCL, AGO, and RDR in sunflower based on bioinformatics approaches. Therefore, we accomplished an inclusive in silico investigation for genome-wide identification of RNAi pathway gene families DCL, AGO, and RDR through bioinformatics approaches such as (sequence homogeneity, phylogenetic relationship, gene structure, chromosomal localization, PPIs, GO, sub-cellular localization). In this study, we have identified five DCL (HaDCLs), fifteen AGO (HaAGOs), and ten RDR (HaRDRs) in the sunflower genome database corresponding to the RNAi genes of model plant Arabidopsis thaliana based on genome-wide analysis and a phylogenetic method. The analysis of the gene structure that contains exon-intron numbers, conserved domain, and motif composition analyses for all HaDCL, HaAGO, and HaRDR gene families indicated almost homogeneity among the same gene family. The protein-protein interaction (PPI) network analysis illustrated that there exists interconnection among identified three gene families. The analysis of the Gene Ontology (GO) enrichment showed that the detected genes directly contribute to the RNA gene-silencing and were involved in crucial pathways. It was observed that the cis-acting regulatory components connected to the identified genes were shown to be responsive to hormone, light, stress, and other functions. That was found in HaDCL, HaAGO, and HaRDR genes associated with the development and growth of plants. Finally, we are able to provide some essential information about the components of sunflower RNA silencing through our genome-wide comparison and integrated bioinformatics analysis, which open the door for further research into the functional mechanisms of the identified genes and their regulatory elements.

Genome-wide analysis of key gene families in RNA silencing and their responses to biotic and drought stresses in adzuki bean

BackgroundIn plants, RNA silencing is an important conserved mechanism to regulate gene expression and combat against abiotic and biotic stresses. Dicer-like (DCL) and Argonaute (AGO) proteins and RNA-dependent RNA polymerase (RDR) are the core elements involved in gene silencing and their gene families have been explored in many plants. However, these genes and their responses to stresses have not yet been well characterized in adzuki bean.ResultsA total of 11 AGO, 7 DCL and 6 RDR proteins were identified, and phylogenetic analyses of these proteins showed that they clustered into six, four and four clades respectively. The expression patterns of these genes in susceptible or resistant adzuki bean cultivars challenged with drought, bean common mosaic virus and Podosphaera xanthii infections were further validated by quantitative RT-PCR. The different responses of these proteins under abiotic and biotic stresses indicated their specialized regulatory mechanisms.ConclusionsIn this study, 24 genes of the DCL, AGO and RDR gene families in adzuki bean were identified, and the sequence characterization, structure of the encoded proteins, evolutionary relationship with orthologues in other legumes and gene expression patterns under drought and biotic stresses were primarily explored, which enriched our understanding of these genes in adzuki bean. Our findings provide a foundation for the comparative genomic analyses of RNA silencing elements in legume plants and further new insights into the functional complexity of RNA silencing in the response to various stresses in adzuki bean.

Genome-Wide Identification and Characterization of Argonaute, Dicer-like and RNA-Dependent RNA Polymerase Gene Families and Their Expression Analyses in Fragaria spp.

In the growth and development of plants, some non-coding small RNAs (sRNAs) not only mediate RNA interference at the post-transcriptional level, but also play an important regulatory role in chromatin modification at the transcriptional level. In these processes, the protein factors Argonaute (AGO), Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) play very important roles in the synthesis of sRNAs respectively. Though they have been identified in many plants, the information about these gene families in strawberry was poorly understood. In this study, using a genome-wide analysis and a phylogenetic approach, 13 AGO, six DCL, and nine RDR genes were identified in diploid strawberry Fragaria vesca. We also identified 33 AGO, 18 DCL, and 28 RDR genes in octoploid strawberry Fragaria × ananassa, studied the expression patterns of these genes in various tissues and developmental stages of strawberry, and researched the response of these genes to some hormones, finding that almost all genes respond to the five hormone stresses. This study is the first report of a genome-wide analysis of AGO, DCL, and RDR gene families in Fragaria spp., in which we provide basic genomic information and expression patterns for these genes. Additionally, this study provides a basis for further research on the functions of these genes and some evidence for the evolution between diploid and octoploid strawberries.

Genome-wide identification of DCL, AGO and RDR gene families and their associated functional regulatory elements analyses in banana (Musa acuminata).

RNA silencing is mediated through RNA interference (RNAi) pathway gene families, i.e., Dicer-Like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) and their cis-acting regulatory elements. The RNAi pathway is also directly connected with the post-transcriptional gene silencing (PTGS) mechanism, and the pathway controls eukaryotic gene regulation during growth, development, and stress response. Nevertheless, genome-wide identification of RNAi pathway gene families such as DCL, AGO, and RDR and their regulatory network analyses related to transcription factors have not been studied in many fruit crop species, including banana (Musa acuminata). In this study, we studied in silico genome-wide identification and characterization of DCL, AGO, and RDR genes in bananas thoroughly via integrated bioinformatics approaches. A genome-wide analysis identified 3 MaDCL, 13 MaAGO, and 5 MaRDR candidate genes based on multiple sequence alignment and phylogenetic tree related to the RNAi pathway in banana genomes. These genes correspond to the Arabidopsis thaliana RNAi silencing genes. The analysis of the conserved domain, motif, and gene structure (exon-intron numbers) for MaDCL, MaAGO, and MaRDR genes showed higher homogeneity within the same gene family. The Gene Ontology (GO) enrichment analysis exhibited that the identified RNAi genes could be involved in RNA silencing and associated metabolic pathways. A number of important transcription factors (TFs), e.g., ERF, Dof, C2H2, TCP, GATA and MIKC_MADS families, were identified by network and sub-network analyses between TFs and candidate RNAi gene families. Furthermore, the cis-acting regulatory elements related to light-responsive (LR), stress-responsive (SR), hormone-responsive (HR), and other activities (OT) functions were identified in candidate MaDCL, MaAGO, and MaRDR genes. These genome-wide analyses of these RNAi gene families provide valuable information related to RNA silencing, which would shed light on further characterization of RNAi genes, their regulatory elements, and functional roles, which might be helpful for banana improvement in the breeding program.

In silico identification and characterization of AGO, DCL and RDR gene families and their associated regulatory elements in sweet orange (Citrus sinensis L.)

RNA interference (RNAi) plays key roles in post-transcriptional and chromatin modification levels as well as regulates various eukaryotic gene expressions which are involved in stress responses, development and maintenance of genome integrity during developmental stages. The whole mechanism of RNAi pathway is directly involved with the gene-silencing process by the interaction of Dicer-Like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) gene families and their regulatory elements. However, these RNAi gene families and their sub-cellular locations, functional pathways and regulatory components were not extensively investigated in the case of economically and nutritionally important fruit plant sweet orange (Citrus sinensis L.). Therefore, in silico characterization, gene diversity and regulatory factor analysis of RNA silencing genes in C. sinensis were conducted by using the integrated bioinformatics approaches. Genome-wide comparison analysis based on phylogenetic tree approach detected 4 CsDCL, 8 CsAGO and 4 CsRDR as RNAi candidate genes in C. sinensis corresponding to the RNAi genes of model plant Arabidopsis thaliana. The domain and motif composition and gene structure analyses for all three gene families exhibited almost homogeneity within the same group members. The Gene Ontology enrichment analysis clearly indicated that the predicted genes have direct involvement into the gene-silencing and other important pathways. The key regulatory transcription factors (TFs) MYB, Dof, ERF, NAC, MIKC_MADS, WRKY and bZIP were identified by their interaction network analysis with the predicted genes. The cis-acting regulatory elements associated with the predicted genes were detected as responsive to light, stress and hormone functions. Furthermore, the expressed sequence tag (EST) analysis showed that these RNAi candidate genes were highly expressed in fruit and leaves indicating their organ specific functions. Our genome-wide comparison and integrated bioinformatics analyses provided some necessary information about sweet orange RNA silencing components that would pave a ground for further investigation of functional mechanism of the predicted genes and their regulatory factors.

Genome-wide identification and characterization of DCL, AGO and RDR gene families in Saccharum spontaneum

RNA silencing is a conserved mechanism in eukaryotic organisms to regulate gene expression. Argonaute (AGO), Dicer-like (DCL) and RNA-dependent RNA polymerase (RDR) proteins are critical components of RNA silencing, but how these gene families’ functions in sugarcane were largely unknown. Most stress-resistance genes in modern sugarcane cultivars (Saccharum spp.) were originated from wild species of Saccharum, for example S. spontaneum. Here, we used genome-wide analysis and a phylogenetic approach to identify four DCL, 21 AGO and 11 RDR genes in the S. spontaneum genome (termed SsDCL, SsAGO and SsRDR, respectively). Several genes, particularly some of the SsAGOs, appeared to have undergone tandem or segmental duplications events. RNA-sequencing data revealed that four SsAGO genes (SsAGO18c, SsAGO18b, SsAGO10e and SsAGO6b) and three SsRDR genes (SsRDR2b, SsRDR2d and SsRDR3) tended to have preferential expression in stem tissue, while SsRDR5 was preferentially expressed in leaves. qRT-PCR analysis showed that SsAGO10c, SsDCL2 and SsRDR6b expressions were strongly upregulated, whereas that of SsAGO18b, SsRDR1a, SsRDR2b/2d and SsRDR5 was significantly depressed in S. spontaneum plants exposed to PEG-induced dehydration stress or infected with Xanthomonas albilineans, causal agent of leaf scald disease of sugarcane, suggesting that these genes play important roles in responses of S. spontaneum to biotic and abiotic stresses.

Genome-wide analysis of AGO, DCL and RDR gene families reveals RNA-directed DNA methylation is involved in fruit abscission in Citrus sinensis

BackgroundSmall RNAs regulate a wide variety of processes in plants, from organ development to both biotic and abiotic stress response. Being master regulators in genetic networks, their biogenesis and action is a fundamental aspect to characterize in order to understand plant growth and development. Three main gene families are critical components of RNA silencing: DICER-LIKE (DCL), ARGONAUTE (AGO) and RNA-DEPENDENT RNA POLYMERASE (RDR). Even though they have been characterized in other plant species, there is no information about these gene families in Citrus sinensis, one of the most important fruit species from both economical and nutritional reasons. While small RNAs have been implicated in the regulation of multiple aspects of plant growth and development, their role in the abscission process has not been characterized yet.ResultsUsing genome-wide analysis and a phylogenetic approach, we identified a total of 13 AGO, 5 DCL and 7 RDR genes. We characterized their expression patterns in root, leaf, flesh, peel and embryo samples using RNA-seq data. Moreover, we studied their role in fruit abscission through gene expression analysis in fruit rind compared to abscission zone from samples obtained by laser capture microdissection. Interestingly, we determined that the expression of several RNA silencing factors are down-regulated in fruit abscission zone, being particularly represented gene components of the RNA-dependent DNA Methylation pathway, indicating that repression of this process is necessary for fruit abscission to take place in Citrus sinensis.ConclusionsThe members of these 3 families present characteristic conserved domains and distinct expression patterns. We provide a detailed analysis of the members of these families and improved the annotation of some of these genes based on RNA-seq data. Our data suggests that the RNA-dependent DNA Methylation pathway is involved in the important fruit abscission process in C. sinensis.

Genome-Wide Identification of the Dicer-Like, Argonaute, and RNA-Dependent RNA Polymerase Gene Families in Cucumber (Cucumis sativus L.)

Dicer, Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) comprise the core components of RNA-induced silencing complexes, which trigger RNA silencing. Here, we performed a complete analysis of the cucumber Dicer-like, AGO, and RDR gene families including the gene structure, genomic localization, and phylogenetic relationships among family members. We identified seven CsAGO genes, five CsDCL genes, and eight CsRDR genes in cucumber. Based on phylogenetic analysis, each of these genes families was categorized into three or four clades. The orthologs of CsAGOs, CsDCLs, and CsRDRs were identified in apple, peach, wild strawberry, foxtail millet, and maize, and the evolutionary relationships among the orthologous gene pairs were investigated. We also investigated the expression levels of CsAGOs, CsDCLs, and CsRDRs in various cucumber tissues. All CsAGOs were relatively higher upregulated in leaves and tendrils than in other organs, especially CsAGO1c, CsAGO1d, and CsAGO7. All CsDCL genes were relatively higher upregulated in tendrils, with almost no expression detected for CsDCL1, CsDCL4a, or CsDCL4b in other organs. In addition, CsRDR1a, CsRDR2, CsRDR3, and CsRDR6 had relatively higher upregulation in tendrils, whereas almost all CsRDRs were downregulation in other organs. The results of this study will facilitate further studies of gene silencing pathways in cucumber.

Comprehensive Analysis of Dicer-Like, Argonaute, and RNA-dependent RNA Polymerase Gene Families in Grapevine (Vitis Vinifera)

Regulation of gene expression is associated with RNA interference (RNAi) at the posttranscriptional level and chromatin modification in transcriptional silencing during plant development. Dicer-like (DCL) and Argonaute (AGO) proteins, along with RNA-dependent RNA polymerase (RDR), play integral roles in these processes. Many members of the DCL, AGO, and RDR gene families have been characterized in Arabidopsis, rice, maize, and tomato. However, little is known about these genes in grapevine. Here, we implemented a comprehensive bioinformatics analysis of the DCL, AGO, and RDR gene families in grapevine. In this study, a total of four VvDCL, thirteen VvAGO, and five VvRDR genes were identified in grapevine. These genes were categorized into four groups for each family based on phylogenetic analysis. Twenty-two genes were distributed across 11 chromosomes. We detected five segmental or tandem duplication events, which suggested their conserved functions during evolution. In each subfamily, protein motif composition was relatively conserved. The digital EST expression analyses provided a first glimpse of the expression patterns of DCL, AGO, and RDR gene in grapevine. It is worth mentioning that one gene, VvAGO10a, only expressed in the stem, which suggested that VvAGO10a may function in the regulation of siRNAs in the grapevine stem. Gene expression patterns during berry development and under different abiotic stresses were investigated utilizing microarray data. Thus, these detailed investigations provide a foundation for transcriptional silencing-associated gene families in grapevine for future functional genomics studies.

Identification, Characterization and Expression Profiling of Dicer-Like, Argonaute and RNA-Dependent RNA Polymerase Gene Families in Foxtail Millet

Post-transcriptional control of gene expression is achieved through RNA interference where the activities of Dicer-like (DCL), Argonautes (AGO) and RNA-dependent RNA polymerases (RDRs) are significant. Hence, considering the importance of DCL, AGO and RDRs, a comprehensive genome-wide analysis was performed in foxtail millet. The study identified 8 DCL, 19 AGO and 11 RDR genes. Phylogenetic and domain analysis provided interesting information on the evolutionary and structural aspects of these proteins. The orthologs of Setaria italica DCL (SiDCL), AGO (SiAGO) and RDRs (SiRDRs) were identified in sorghum, maize and rice, and the evolutionary relationships among the orthologous gene pairs were investigated. Promoter analysis of SiDCL, SiAGO and SiRDR genes revealed the presence of unique and common cis-acting elements at the upstream of respective gene sequences, which serves as binding sites for several developmental and stress-related transcription factors. In silico expression profiling using RNA-sequence data showed tissue-specific expression patterns of these genes in foxtail millet. Candidate genes representing each sub-family were chosen for expression analysis through quantitative real-time PCR (qRT-PCR) under salinity, dehydration and hormonal treatments. It revealed the differential expression pattern of candidate genes at different time points of stresses. This is the first report on genome-wide analysis of SiDCL, SiAGO and SiRDR gene families in foxtail millet, which provides basic genomic information and insights into the putative roles of these genes in abiotic stresses. The present study will serve as a base for further functional characterization of these gene families in foxtail millet and related grass species.

Genome-wide identification of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families and their expression analyses in response to viral infection and abiotic stresses in Solanum lycopersicum

Dicer, Argonaute and RNA-dependent RNA polymerase form the core components to trigger RNA silencing. Although tomato (Solanum lycopersicum) is a dicotyledon model plant, no systematic analysis and expression profiling of these genes in tomato has been undertaken previously. In this study, seven Dicer-like (SlDCLs), 15 Argonaute (SlAGOs) and six RNA-dependent RNA polymerase (SlRDRs) genes were identified in tomato. These genes were categorized into four subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different tissues and organs using online data and semi-quantitative RT-PCR. Many of the candidate genes were up-regulated in response to Tomato yellow leaf curl virus infection and abiotic stresses. The expression models of tandem gene duplications among SlDCL2s indicated the DCL2 family plays an important role in the evolution of tomato.

Identification and characterization of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families in maize

Eukaryotic gene expression is regulated at least by two processes, RNA interference at the post-transcriptional level and chromatin modification at the transcriptional level. Distinct small RNAs (approximately 21-24 nucleotides; sRNAs) were demonstrated to play vital roles in facilitating gene silencing. In plants, the generation of these sRNAs mainly depends on some proteins encoded by respective Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerases (RDR) gene families. Here, we analyzed the DCL, AGO and RDR gene families in maize, including gene structure, phylogenetic relationships, protein conserved motifs and genomic localization among gene family members. A total of 5 Zmdcl, 18 Zmago and 5 Zmrdr genes were identified in maize. Phylogenetic analyses clustered each of these genes families into four subfamilies. In addition, gene chromosomal localization revealed that five pairs of Zmago genes resulted from tandem or segmental duplication, respectively. EST expression data mining revealed that these newly identified genes had temporal and spatial expression pattern. Furthermore, the transcripts of these genes were detected in the leaves by two different abiotic stress treatments using semi-quantitative RT-PCR. The data demonstrated that these genes exhibited different expression levels in stress treatments. The results of this study provided basic genomic information for these gene families and insights into the probable roles of these genes in plant growth and development. This will further provide a solid foundation for future functional genomics studies of Dicer-like, Argonaute and RDR gene families in maize.

Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA Polymerase gene families and their expression analysis during reproductive development and stress in rice

BackgroundImportant developmental processes in both plants and animals are partly regulated by genes whose expression is modulated at the post-transcriptional level by processes such as RNA interference (RNAi). Dicers, Argonautes and RNA-dependent RNA polymerases (RDR) form the core components that facilitate gene silencing and have been implicated in the initiation and maintenance of the trigger RNA molecules, central to process of RNAi. Investigations in eukaryotes have revealed that these proteins are encoded by variable number of genes with plants showing relatively higher number in each gene family. To date, no systematic expression profiling of these genes in any of the organisms has been reported.ResultsIn this study, we provide a complete analysis of rice Dicer-like, Argonaute and RDR gene families including gene structure, genomic localization and phylogenetic relatedness among gene family members. We also present microarray-based expression profiling of these genes during 14 stages of reproductive and 5 stages of vegetative development and in response to cold, salt and dehydration stress. We have identified 8 Dicer-like (OsDCLs), 19 Argonaute (OsAGOs) and 5 RNA-dependent RNA polymerase (OsRDRs) genes in rice. Based on phylogeny, each of these genes families have been categorized into four subgroups. Although most of the genes express both in vegetative and reproductive organs, 2 OsDCLs, 14 OsAGOs and 3 OsRDRs were found to express specifically/preferentially during stages of reproductive development. Of these, 2 OsAGOs exhibited preferential up-regulation in seeds. One of the Argonautes (OsAGO2) also showed specific up-regulation in response to cold, salt and dehydration stress.ConclusionThis investigation has identified 23 rice genes belonging to DCL, Argonaute and RDR gene families that could potentially be involved in reproductive development-specific gene regulatory mechanisms. These data provide an insight into probable domains of activity of these genes and a basis for further, more detailed investigations aimed at understanding the contribution of individual components of RNA silencing machinery during reproductive phase of plant development.