Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungusPecoramyces ruminantiumstrain C1A

PeerJ ◽

10.7717/peerj.4276 ◽

2018 ◽

Vol 6 ◽

pp. e4276 ◽

Cited By ~ 9

Author(s):

Shelby S. Calkins ◽

Nicole C. Elledge ◽

Katherine E. Mueller ◽

Stephen M. Marek ◽

MB Couger ◽

...

Keyword(s):

Rna Interference ◽

Gene Silencing ◽

Neurospora Crassa ◽

Dna Helicase ◽

Small Interfering Rnas ◽

Gene Encoding ◽

Interacting Protein ◽

Genes Encoding ◽

Lactate Levels ◽

Electron Sink

Members of the anaerobic gut fungi (AGF) reside in rumen, hindgut, and feces of ruminant and non-ruminant herbivorous mammals and reptilian herbivores. No protocols for gene insertion, deletion, silencing, or mutation are currently available for the AGF, rendering gene-targeted molecular biological manipulations unfeasible. Here, we developed and optimized an RNA interference (RNAi)-based protocol for targeted gene silencing in the anaerobic gut fungusPecoramyces ruminantiumstrain C1A. Analysis of the C1A genome identified genes encoding enzymes required for RNA silencing in fungi (Dicer, Argonaute,Neurospora crassaQDE-3 homolog DNA helicase, Argonaute-interacting protein, andNeurospora crassaQIP homolog exonuclease); and the competency of C1A germinating spores for RNA uptake was confirmed using fluorescently labeled small interfering RNAs (siRNA). Addition of chemically-synthesized siRNAs targeting D-lactate dehydrogenase (ldhD) gene to C1A germinating spores resulted in marked target gene silencing; as evident by significantly lowerldhDtranscriptional levels, a marked reduction in the D-LDH specific enzymatic activity in intracellular protein extracts, and a reduction in D-lactate levels accumulating in the culture supernatant. Comparative transcriptomic analysis of untreated versus siRNA-treated cultures identified a few off-target siRNA-mediated gene silencing effects. As well, significant differential up-regulation of the gene encoding NAD-dependent 2-hydroxyacid dehydrogenase (Pfam00389) in siRNA-treated C1A cultures was observed, which could possibly compensate for loss of D-LDH as an electron sink mechanism in C1A. The results demonstrate the feasibility of RNAi in anaerobic fungi, and opens the door for gene silencing-based studies in this fungal clade.

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Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungus Pecoramyces ruminantium strain C1A

10.7287/peerj.preprints.3177 ◽

2017 ◽

Author(s):

Shelby S Calkins ◽

Nicole C Elledge ◽

Stephen M. Marek ◽

M B. Couger ◽

Mostafa S Elshahed ◽

...

Keyword(s):

Rna Interference ◽

Gene Silencing ◽

Neurospora Crassa ◽

Dna Helicase ◽

Small Interfering Rnas ◽

Gene Encoding ◽

Interacting Protein ◽

Genes Encoding ◽

Lactate Levels ◽

Electron Sink

Members of the anaerobic gut fungi (AGF) reside in rumen, hindgut, and feces of ruminant and non-ruminant herbivorous mammals and reptilian herbivores. No protocols for gene insertion, deletion, silencing, or mutation are currently available for the AGF, rendering gene-targeted molecular biological manipulations unfeasible. Here, we developed and optimized an RNA interference (RNAi)-based protocol for targeted gene silencing in the anaerobic gut fungus Pecoramyces ruminantium strain C1A. Analysis of the C1A genome identified genes encoding enzymes required for RNA silencing in fungi (Dicer, Argonaute, Neurospora crassa QDE-3 homolog DNA helicase, Argonaute-interacting protein, and Neurospora crassa QIP homolog exonuclease); and the competency of C1A germinating spores for RNA uptake was confirmed using fluorescently labeled small interfering RNAs (siRNA). Addition of chemically-synthesized siRNAs targeting D-lactate dehydrogenase (ldhD) gene to C1A germinating spores resulted in marked target gene silencing; as evident by significantly lower ldhD transcriptional levels, a marked reduction in the D-LDH specific enzymatic activity in intracellular protein extracts, and a reduction in D-lactate levels accumulating in the culture supernatant. Comparative transcriptomic analysis of untreated versus siRNA-treated cultures identified a few off-target siRNA-mediated gene silencing effects. As well, significant differential up-regulation of the gene encoding NAD-dependent 2-hydroxyacid dehydrogenase (Pfam00389) in siRNA-treated C1A cultures was observed, which could possibly compensate for loss of D-LDH as an electron sink mechanism in C1A. The results demonstrate the feasibility of RNAi in anaerobic fungi, and opens the door for gene silencing-based studies in this fungal clade.

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Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungus Pecoramyces ruminantium strain C1A

10.7287/peerj.preprints.3177v1 ◽

2017 ◽

Author(s):

Shelby S Calkins ◽

Nicole C Elledge ◽

Stephen M. Marek ◽

M B. Couger ◽

Mostafa S Elshahed ◽

...

Keyword(s):

Rna Interference ◽

Gene Silencing ◽

Neurospora Crassa ◽

Dna Helicase ◽

Small Interfering Rnas ◽

Gene Encoding ◽

Interacting Protein ◽

Genes Encoding ◽

Lactate Levels ◽

Electron Sink

Members of the anaerobic gut fungi (AGF) reside in rumen, hindgut, and feces of ruminant and non-ruminant herbivorous mammals and reptilian herbivores. No protocols for gene insertion, deletion, silencing, or mutation are currently available for the AGF, rendering gene-targeted molecular biological manipulations unfeasible. Here, we developed and optimized an RNA interference (RNAi)-based protocol for targeted gene silencing in the anaerobic gut fungus Pecoramyces ruminantium strain C1A. Analysis of the C1A genome identified genes encoding enzymes required for RNA silencing in fungi (Dicer, Argonaute, Neurospora crassa QDE-3 homolog DNA helicase, Argonaute-interacting protein, and Neurospora crassa QIP homolog exonuclease); and the competency of C1A germinating spores for RNA uptake was confirmed using fluorescently labeled small interfering RNAs (siRNA). Addition of chemically-synthesized siRNAs targeting D-lactate dehydrogenase (ldhD) gene to C1A germinating spores resulted in marked target gene silencing; as evident by significantly lower ldhD transcriptional levels, a marked reduction in the D-LDH specific enzymatic activity in intracellular protein extracts, and a reduction in D-lactate levels accumulating in the culture supernatant. Comparative transcriptomic analysis of untreated versus siRNA-treated cultures identified a few off-target siRNA-mediated gene silencing effects. As well, significant differential up-regulation of the gene encoding NAD-dependent 2-hydroxyacid dehydrogenase (Pfam00389) in siRNA-treated C1A cultures was observed, which could possibly compensate for loss of D-LDH as an electron sink mechanism in C1A. The results demonstrate the feasibility of RNAi in anaerobic fungi, and opens the door for gene silencing-based studies in this fungal clade.

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Gene Silencing by RNA Interference and the Role of Small Interfering RNAs

Gene Silencing by RNA Interference ◽

10.1201/9780203489253-7 ◽

2004 ◽

pp. 21-34

Keyword(s):

Rna Interference ◽

Gene Silencing ◽

Small Interfering Rnas

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Tobacco rattle virus–induced gene silencing in Hevea brasiliensis

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa085 ◽

2021 ◽

Vol 85 (3) ◽

pp. 562-567

Author(s):

Hui-Liang Li ◽

Dong Guo ◽

Ying Wang ◽

Jia-Hong Zhu ◽

Long Qu ◽

...

Keyword(s):

Gene Silencing ◽

Hevea Brasiliensis ◽

Rubber Tree ◽

Tobacco Rattle Virus ◽

Reverse Genetic ◽

Small Interfering Rnas ◽

Virus Induced Gene Silencing ◽

Gene Encoding ◽

Efficient Gene ◽

Unknown Gene

ABSTRACT Virus-induced gene silencing (VIGS) is a powerful gene-silencing tool that has been intensively applied in plants. To data, the application of VIGS in rubber tree has not yet been reported. In this study, we described the efficient gene silencing in rubber tree by VIGS. The gene encoding Hevea brasiliensis phytoene desaturase (HbPDS) was identified in rubber tree genome. Small interfering RNAs from HbPDS and the silencing gene fragment were predicted and a length of 399 bp was selected to be tested. We showed that the tobacco rattle virus (TRV)-VIGS could induce effective HbPDS silencing in rubber tree. This study was the first to report VIGS in rubber tree. The present TRV-VIGS method could be used to perform reverse genetic approaches to identify unknown gene functions and might be further applied to produce gene silenced rubber tree plants, to advance functional gene of rubber tree.

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Exploring the functions of RNA interference pathway proteins: some functions are more RISCy than others?

Biochemical Journal ◽

10.1042/bj20041822 ◽

2005 ◽

Vol 387 (3) ◽

pp. 561-571 ◽

Cited By ~ 32

Author(s):

Katarzyna JARONCZYK ◽

Jon B. CARMICHAEL ◽

Tom C. HOBMAN

Keyword(s):

Rna Interference ◽

Gene Silencing ◽

Cell Cycle Regulators ◽

Small Interfering Rnas ◽

Piwi Domain ◽

Therapeutic Technique ◽

Silencing Pathways ◽

Laboratory Tool ◽

Molecular Therapeutic ◽

Regulate Gene

PPD (PAZ Piwi domain) proteins and the Dicer family have been the subjects of intense study over the last 6 years. These proteins have well-established roles in RNAi (RNA interference), a process that relies on siRNAs (small interfering RNAs) or miRNAs (microRNAs) to mediate specificity. The development of techniques for applying RNAi as a laboratory tool and a molecular therapeutic technique has rapidly outpaced our understanding of the biology of this process. However, over the last 2 years, great strides have been made towards elucidating how PPD proteins and Dicer regulate gene-silencing at the pre- and post-transcriptional levels. In addition, evidence is beginning to emerge that suggests that these proteins have additional siRNA-independent roles as cell-cycle regulators. In the present review, we summarize the well-known roles of these two classes of proteins in gene-silencing pathways, as well as explore the evidence for novel roles of PPD and Dicer proteins.

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Cross-Kingdom Small RNAs Among Animals, Plants and Microbes

Cells ◽

10.3390/cells8040371 ◽

2019 ◽

Vol 8 (4) ◽

pp. 371 ◽

Cited By ~ 20

Author(s):

Zeng ◽

Gupta ◽

Jiang ◽

Yang ◽

Gong ◽

...

Keyword(s):

Gene Expression ◽

Rna Interference ◽

Gene Silencing ◽

Horizontal Transfer ◽

Small Rnas ◽

Signal Transmission ◽

Fungal Diseases ◽

Small Interfering Rnas ◽

Eukaryotic Species ◽

Non Coding Rnas

Small RNAs (sRNAs), a class of regulatory non-coding RNAs around 20~30-nt long, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), are critical regulators of gene expression. Recently, accumulating evidence indicates that sRNAs can be transferred not only within cells and tissues of individual organisms, but also across different eukaryotic species, serving as a bond connecting the animal, plant, and microbial worlds. In this review, we summarize the results from recent studies on cross-kingdom sRNA communication. We not only review the horizontal transfer of sRNAs among animals, plants and microbes, but also discuss the mechanism of RNA interference (RNAi) signal transmission via cross-kingdom sRNAs. We also compare the advantages of host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS) technology and look forward to their applicable prospects in controlling fungal diseases.

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Small Interfering RNAs That Trigger Posttranscriptional Gene Silencing Are Not Required for the Histone H3 Lys9 Methylation Necessary for Transgenic Tandem Repeat Stabilization in Neurospora crassa

Molecular and Cellular Biology ◽

10.1128/mcb.25.9.3793-3801.2005 ◽

2005 ◽

Vol 25 (9) ◽

pp. 3793-3801 ◽

Cited By ~ 36

Author(s):

Agustin Chicas ◽

Emma C. Forrest ◽

Silvia Sepich ◽

Carlo Cogoni ◽

Giuseppe Macino

Keyword(s):

Gene Silencing ◽

Neurospora Crassa ◽

Chromatin Immunoprecipitation ◽

Histone H3 ◽

Transcriptional Gene Silencing ◽

Small Interfering Rnas ◽

Posttranscriptional Gene Silencing ◽

Endogenous Gene ◽

Homologous Genes ◽

Interfering Rna

ABSTRACT In Neurospora crassa, the introduction of a transgene can lead to small interfering RNA (siRNA)-mediated posttranscriptional gene silencing (PTGS) of homologous genes. siRNAs can also guide locus-specific methylation of Lys9 of histone H3 (Lys9H3) in Schizosaccharomyces pombe. Here we tested the hypothesis that transgenically derived siRNAs may contemporaneously both activate the PTGS mechanism and induce chromatin modifications at the transgene and the homologous endogenous gene. We carried out chromatin immunoprecipitation using a previously characterized albino-1 (al-1) silenced strain but detected no alterations in the pattern of histone modifications at the endogenous al-1 locus, suggesting that siRNAs produced from the transgenic locus do not trigger modifications in trans of those histones tested. Instead, we found that the transgenic locus was hypermethylated at Lys9H3 in our silenced strain and remained hypermethylated in the quelling defective mutants (qde), further demonstrating that the PTGS machinery is dispensable for Lys9H3 methylation. However, we found that a mutant in the histone Lys9H3 methyltransferase dim-5 was unable to maintain PTGS, with transgenic copies being rapidly lost, resulting in reversion of the silenced phenotype. These results indicate that the defect in PTGS of the Δdim-5 strain is due to the inability to maintain the transgene in tandem, suggesting a role for DIM-5 in stabilizing such repeated sequences. We conclude that in Neurospora, siRNAs produced from the transgenic locus are used in the RNA-induced silencing complex-mediated PTGS pathway and do not communicate with an RNAi-induced initiation of transcriptional gene silencing complex to effect chromatin-based silencing.

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Chemical strategies for strand selection in short-interfering RNAs

RSC Advances ◽

10.1039/d0ra07747j ◽

2021 ◽

Vol 11 (4) ◽

pp. 2415-2426

Author(s):

Andrew J. Varley ◽

Jean-Paul Desaulniers

Keyword(s):

Rna Interference ◽

Gene Silencing ◽

Rnai Pathway ◽

Specific Gene ◽

Small Interfering Rnas ◽

Chemical Strategies

Therapeutic small interfering RNAs (siRNAs) are double stranded RNAs capable of potent and specific gene silencing through activation of the RNA interference (RNAi) pathway.

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