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

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Huimin Meng ◽  
Zhangxun Wang ◽  
Yulong Wang ◽  
Hong Zhu ◽  
Bo Huang
Keyword(s):  
Rna Interference ◽  
Entomopathogenic Fungus ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Digital Gene Expression ◽  
Abiotic Stress Response ◽  
Mycelium Growth ◽  
Metarhizium Robertsii ◽  
Content Type ◽  
Genes Encoding

ABSTRACT 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. robertsii dcl1 [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. robertsii. IMPORTANCE 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.

scholarly journals Mr-AbaA Regulates Conidiation by Interacting with the Promoter Regions of Both Mr-veA and Mr-wetA in Metarhizium robertsii

2021 ◽  
Author(s):  
Hao Wu ◽  
Youmin Tong ◽  
Rong Zhou ◽  
Yulong Wang ◽  
Zhangxun Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Entomopathogenic Fungus ◽  
Submerged Culture ◽  
Comprehensive Understanding ◽  
Promoter Regions ◽  
Metarhizium Robertsii ◽  
Content Type

Metarhizium robertsii is an emerging model entomopathogenic fungus for developing biopesticides; therefore, a comprehensive understanding of its conidiation is very important for its application. In this study, we revealed that the transcription factor Mr-AbaA is involved in the control of aerial conidiation and blastospore separation in submerged culture.

scholarly journals Unveiling of the Diversity of Prasinoviruses (Phycodnaviridae) in Marine Samples by Using High-Throughput Sequencing Analyses of PCR-Amplified DNA Polymerase and Major Capsid Protein Genes

2014 ◽  
Vol 80 (10) ◽  
pp. 3150-3160 ◽  
Author(s):  
Camille Clerissi ◽  
Nigel Grimsley ◽  
Hiroyuki Ogata ◽  
Pascal Hingamp ◽  
Julie Poulain ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Dna Polymerase ◽  
High Throughput Sequencing ◽  
Major Capsid Protein ◽  
Content Type ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Genes Encoding ◽  
Conserved Genes ◽  
Primer Sets

ABSTRACTViruses strongly influence the ecology and evolution of their eukaryotic hosts in the marine environment, but little is known about their diversity and distribution. Prasinoviruses infect an abundant and widespread class of phytoplankton, the Mamiellophyceae, and thereby exert a specific and important role in microbial ecosystems. However, molecular tools to specifically identify this viral genus in environmental samples are still lacking. We developed two primer sets, designed for use with polymerase chain reactions and 454 pyrosequencing technologies, to target two conserved genes, encoding the DNA polymerase (PolB gene) and the major capsid protein (MCP gene). While only one copy of the PolB gene is present inPrasinovirusgenomes, there are at least seven paralogs for MCP, the copy we named number 6 being shared with other eukaryotic alga-infecting viruses. Primer sets for PolB and MCP6 were thus designed and tested on 6 samples from the Tara Oceans project. The results suggest that the MCP6 amplicons show greater richness but that PolB gave a wider coverage ofPrasinovirusdiversity. As a consequence, we recommend use of the PolB primer set, which will certainly reveal exciting new insights about the diversity and distribution of prasinoviruses at the community scale.

scholarly journals Identification of Mycoparasitism-Related Genes in Trichoderma atroviride

2011 ◽  
Vol 77 (13) ◽  
pp. 4361-4370 ◽  
Author(s):  
Barbara Reithner ◽  
Enrique Ibarra-Laclette ◽  
Robert L. Mach ◽  
Alfredo Herrera-Estrella
Keyword(s):  
High Throughput Sequencing ◽  
Pathogenic Fungus ◽  
Phytophthora Capsici ◽  
Expression Patterns ◽  
Trichoderma Atroviride ◽  
Acetyl Xylan Esterase ◽  
Promoter Regions ◽  
Content Type ◽  
Genes Encoding ◽  
Differential Transcription

ABSTRACTA high-throughput sequencing approach was utilized to carry out a comparative transcriptome analysis ofTrichoderma atrovirideIMI206040 during mycoparasitic interactions with the plant-pathogenic fungusRhizoctonia solani. In this study, transcript fragments of 7,797Trichodermagenes were sequenced, 175 of which were host responsive. According to the functional annotation of these genes by KOG (eukaryotic orthologous groups), the most abundant group during direct contact was “metabolism.” Quantitative reverse transcription (RT)-PCR confirmed the differential transcription of 13 genes (includingswo1, encoding an expansin-like protein;axe1, coding for an acetyl xylan esterase; and homologs of genes encoding the aspartyl proteasepapAand a trypsin-like protease,pra1) in the presence ofR. solani. An additional relative gene expression analysis of these genes, conducted at different stages of mycoparasitism againstBotrytis cinereaandPhytophthora capsici, revealed a synergistic transcription of various genes involved in cell wall degradation. The similarities in expression patterns and the occurrence of regulatory binding sites in the corresponding promoter regions suggest a possible analog regulation of these genes during the mycoparasitism ofT. atroviride. Furthermore, a chitin- and distance-dependent induction ofpra1was demonstrated.

scholarly journals RNAi-Mediated Resistance Against Viruses in Perennial Fruit Plants

Plants ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 359 ◽  
Author(s):  
Khushwant Singh ◽  
Chris Dardick ◽  
Jiban Kumar Kundu
Keyword(s):  
Rna Interference ◽  
Stress Responses ◽  
Small Rnas ◽  
Messenger Rna ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Current Knowledge ◽  
Plant Immunity ◽  
Plant Viruses ◽  
Perennial Plants

Small RNAs (sRNAs) are 20–30-nucleotide-long, regulatory, noncoding RNAs that induce silencing of target genes at the transcriptional and posttranscriptional levels. They are key components for cellular functions during plant development, hormone signaling, and stress responses. Generated from the cleavage of double-stranded RNAs (dsRNAs) or RNAs with hairpin structures by Dicer-like proteins (DCLs), they are loaded onto Argonaute (AGO) protein complexes to induce gene silencing of their complementary targets by promoting messenger RNA (mRNA) cleavage or degradation, translation inhibition, DNA methylation, and/or histone modifications. This mechanism of regulating RNA activity, collectively referred to as RNA interference (RNAi), which is an evolutionarily conserved process in eukaryotes. Plant RNAi pathways play a fundamental role in plant immunity against viruses and have been exploited via genetic engineering to control disease. Plant viruses of RNA origin that contain double-stranded RNA are targeted by the RNA-silencing machinery to produce virus-derived small RNAs (vsRNAs). Some vsRNAs serve as an effector to repress host immunity by capturing host RNAi pathways. High-throughput sequencing (HTS) strategies have been used to identify endogenous sRNA profiles, the “sRNAome”, and analyze expression in various perennial plants. Therefore, the review examines the current knowledge of sRNAs in perennial plants and fruits, describes the development and implementation of RNA interference (RNAi) in providing resistance against economically important viruses, and explores sRNA targets that are important in regulating a variety of biological processes.

scholarly journals CytochromebdOxidase Has an Important Role in Sustaining Growth and Development ofStreptomyces coelicolorA3(2) under Oxygen-Limiting Conditions

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Marco Fischer ◽  
Dörte Falke ◽  
Carolin Naujoks ◽  
R. Gary Sawers
Keyword(s):  
Streptomyces Coelicolor ◽  
Reduction Rate ◽  
Growth Conditions ◽  
Developmental Cycle ◽  
Wild Type ◽  
Mycelium Growth ◽  
Developmental Defects ◽  
Content Type ◽  
Mycelia Growth ◽  
Genes Encoding

ABSTRACTStreptomyces coelicolorA3(2) is a filamentously growing, spore-forming, obligately aerobic actinobacterium that uses both a copperaa3-type cytochromecoxidase and a cytochromebdoxidase to respire oxygen. Using defined knockout mutants, we demonstrated that either of these terminal oxidases was capable of allowing the bacterium to grow and complete its developmental cycle. The genes encoding thebcccomplex and theaa3oxidase are clustered at a single locus. Using Western blot analyses, we showed that thebcc-aa3oxidase branch is more prevalent in spores than thebdoxidase. The level of the catalytic subunit, CydA, of thebdoxidase was low in spore extracts derived from the wild type, but it was upregulated in a mutant lacking thebcc-aa3supercomplex. This indicates that cytochromebdoxidase can compensate for the lack of the other respiratory branch. Components of both oxidases were abundant in growing mycelium. Growth studies in liquid medium revealed that a mutant lacking thebcc-aa3oxidase branch grew approximately half as fast as the wild type, while the oxygen reduction rate of the mutant remained close to that of the wild type, indicating that thebdoxidase was mainly functioning in controlling electron flux. Developmental defects were observed for a mutant lacking the cytochromebdoxidase during growth on buffered rich medium plates with glucose as the energy substrate. Evidence based on using the redox-cycling dye methylene blue suggested that cytochromebdoxidase is essential for the bacterium to grow and complete its developmental cycle under oxygen limitation.IMPORTANCERespiring with oxygen is an efficient means of conserving energy in biological systems. The spore-forming, filamentous actinobacteriumStreptomyces coelicolorgrows only aerobically, synthesizing two enzyme complexes for O2reduction, the cytochromebcc-aa3cytochrome oxidase supercomplex and the cytochromebdoxidase. We show in this study that the bacterium can survive with either of these respiratory pathways to oxygen. Immunological studies indicate that thebcc-aa3oxidase is the main oxidase present in spores, but thebdoxidase compensates if thebcc-aa3oxidase is inactivated. Both oxidases are active in mycelia. Growth conditions were identified, revealing that cytochromebdoxidase is essential for aerial hypha formation and sporulation, and this was linked to an important role of the enzyme under oxygen-limiting conditions.

scholarly journals RNA Interference of Genes Encoding the Vacuolar-ATPase in Liriomyza trifolii

Insects ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 41
Author(s):  
Ya-Wen Chang ◽  
Yu-Cheng Wang ◽  
Xiao-Xiang Zhang ◽  
Junaid Iqbal ◽  
Yu-Zhou Du
Keyword(s):  
Rna Interference ◽  
Expression Analysis ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Control Strategies ◽  
Vacuolar Atpase ◽  
Invasive Pest ◽  
Liriomyza Trifolii ◽  
Horticultural Crops ◽  
Genes Encoding

The leafminer fly, Liriomyza trifolii, is an invasive pest of vegetable and horticultural crops in China. In this study, a microinjection method based on dsRNA was developed for RNA interference (RNAi) in L. trifolii using genes encoding vacuolar-ATPase (V-ATPase). Expression analysis indicated that V-ATPase B and V-ATPase D were more highly expressed in L. trifolii adults than in larvae or pupae. Microinjection experiments with dsV-ATPase B and dsV-ATPase D were conducted to evaluate the efficacy of RNAi in L. trifolii adults. Expression analysis indicated that microinjection with 100 ng dsV-ATPase B or dsV-ATPase led to a significant reduction in V-ATPase transcripts as compared to that of the dsGFP control (dsRNA specific to green fluorescent protein). Furthermore, lower dsRNA concentrations were also effective in reducing the expression of target genes when delivered by microinjection. Mortality was significantly higher in dsV-ATPase B- and dsV-ATPase D-treated insects than in controls injected with dsGFP. The successful deployment of RNAi in L. trifolii will facilitate functional analyses of vital genes in this economically-important pest and may ultimately result in new control strategies.

scholarly journals Widespread occurrence of microRNA-mediated target cleavage on membrane-bound polysomes

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xiaoyu Yang ◽  
Chenjiang You ◽  
Xufeng Wang ◽  
Lei Gao ◽  
Beixin Mo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Gene Silencing ◽  
High Frequency ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Noncoding Rnas ◽  
Small Interfering Rnas ◽  
Widespread Occurrence ◽  
Membrane Bound ◽  
Different Tissues

Abstract Background Small RNAs (sRNAs) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) serve as core players in gene silencing at transcriptional and post-transcriptional levels in plants, but their subcellular localization has not yet been well studied, thus limiting our mechanistic understanding of sRNA action. Results We investigate the cytoplasmic partitioning of sRNAs and their targets globally in maize (Zea mays, inbred line “B73”) and rice (Oryza sativa, cv. “Nipponbare”) by high-throughput sequencing of polysome-associated sRNAs and 3′ cleavage fragments, and find that both miRNAs and a subset of 21-nucleotide (nt)/22-nt siRNAs are enriched on membrane-bound polysomes (MBPs) relative to total polysomes (TPs) across different tissues. Most of the siRNAs are generated from transposable elements (TEs), and retrotransposons positively contributed to MBP overaccumulation of 22-nt TE-derived siRNAs (TE-siRNAs) as opposed to DNA transposons. Widespread occurrence of miRNA-mediated target cleavage is observed on MBPs, and a large proportion of these cleavage events are MBP-unique. Reproductive 21PHAS (21-nt phasiRNA-generating) and 24PHAS (24-nt phasiRNA-generating) precursors, which were commonly considered as noncoding RNAs, are bound by polysomes, and high-frequency cleavage of 21PHAS precursors by miR2118 and 24PHAS precursors by miR2275 is further detected on MBPs. Reproductive 21-nt phasiRNAs are enriched on MBPs as opposed to TPs, whereas 24-nt phasiRNAs are nearly completely devoid of polysome occupancy. Conclusions MBP overaccumulation is a conserved pattern for cytoplasmic partitioning of sRNAs, and endoplasmic reticulum (ER)-bound ribosomes function as an independent regulatory layer for miRNA-induced gene silencing and reproductive phasiRNA biosynthesis in maize and rice.

scholarly journals Genes Required for Aerial Growth, Cell Division, and Chromosome Segregation Are Targets of WhiA before Sporulation in Streptomyces venezuelae

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Matthew J. Bush ◽  
Maureen J. Bibb ◽  
Govind Chandra ◽  
Kim C. Findlay ◽  
Mark J. Buttner
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Regulatory Networks ◽  
Liquid Culture ◽  
Biological Processes ◽  
Streptomyces Venezuelae ◽  
Content Type ◽  
Master Regulators ◽  
Aerial Growth ◽  
Genes Encoding

ABSTRACTWhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacteriumStreptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA inStreptomycesdevelopment and its function as a transcription factor, we identified the WhiA regulon through a combination of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray transcriptional profiling, exploiting a new model organism for the genus,Streptomyces venezuelae, which sporulates in liquid culture. The regulon encompasses ~240 transcription units, and WhiA appears to function almost equally as an activator and as a repressor. Bioinformatic analysis of the upstream regions of the complete regulon, combined with DNase I footprinting, identified a short but highly conserved asymmetric sequence, GACAC, associated with the majority of WhiA targets. Construction of a null mutant showed thatwhiAis required for the initiation of sporulation septation and chromosome segregation inS. venezuelae, and several genes encoding key proteins of theStreptomycescell division machinery, such asftsZ,ftsW, andftsK, were found to be directly activated by WhiA during development. Several other genes encoding proteins with important roles in development were also identified as WhiA targets, including the sporulation-specific sigma factor σWhiGand the diguanylate cyclase CdgB. Cell division is tightly coordinated with the orderly arrest of apical growth in the sporogenic cell, andfilP, encoding a key component of the polarisome that directs apical growth, is a direct target for WhiA-mediated repression during sporulation.IMPORTANCESince the initial identification of the genetic loci required forStreptomycesdevelopment, all of thebldandwhidevelopmental master regulators have been cloned and characterized, and significant progress has been made toward understanding the cell biological processes that drive morphogenesis. A major challenge now is to connect the cell biological processes and the developmental master regulators by dissecting the regulatory networks that link the two. Studies of these regulatory networks have been greatly facilitated by the recent introduction ofStreptomyces venezuelaeas a new model system for the genus, a species that sporulates in liquid culture. Taking advantage ofS. venezuelae, we have characterized the regulon of genes directly under the control of one of these master regulators, WhiA. Our results implicate WhiA in the direct regulation of key steps in sporulation, including the cessation of aerial growth, the initiation of cell division, and chromosome segregation.

scholarly journals Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

2014 ◽  
Vol 81 (3) ◽  
pp. 1024-1031 ◽  
Author(s):  
Bhagyalakshmi Kalidass ◽  
Muhammad Farhan Ul-Haque ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
Jeremy D. Semrau
Keyword(s):  
Methane Monooxygenase ◽  
High Specificity ◽  
Copper Uptake ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
Sds Page ◽  
Genes Encoding ◽  
Key Factor ◽  
Membrane Bound

ABSTRACTIt is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that inMethylosinus trichosporiumOB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced byM. trichosporiumOB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and activein situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

scholarly journals Molecular Epidemiology of NDM-1-Producing Enterobacteriaceae and Acinetobacter baumannii Isolates from Pakistan

2014 ◽  
Vol 58 (9) ◽  
pp. 5589-5593 ◽  
Author(s):  
Anna L. Sartor ◽  
Muhammad W. Raza ◽  
Shahid A. Abbasi ◽  
Kathryn M. Day ◽  
John D. Perry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Epidemiology ◽  
Genetic Relatedness ◽  
Antibiotic Resistance Genes ◽  
Enterobacter Cloacae ◽  
Content Type ◽  
Plasmid Replicon ◽  
Genes Encoding ◽  
Clonal Spread ◽  
Encoding Genes

ABSTRACTThe molecular epidemiology of 66 NDM-producing isolates from 2 Pakistani hospitals was investigated, with their genetic relatedness determined using repetitive sequence-based PCR (Rep-PCR). PCR-based replicon typing and screening for antibiotic resistance genes encoding carbapenemases, other β-lactamases, and 16S methylases were also performed. Rep-PCR suggested a clonal spread ofEnterobacter cloacaeandEscherichia coli. A number of plasmid replicon types were identified, with the incompatibility A/C group (IncA/C) being the most common (78%). 16S methylase-encoding genes were coharbored in 81% of NDM-producingEnterobacteriaceae.

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