Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes

Costa, Ana; Mills, Peter R.; Bailey, Andy M.; Foster, Gary D.; Challen, Michael P.

doi:10.1016/j.mimet.2008.06.006

Cited by 18 publications

(13 citation statements)

References 19 publications

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“…Liquid-grown cultures were successfully protoplasted using Trichoderma sp. lysing enzyme, generating in excess of 10 8 protoplasts from a single flask culture. Protoplasts transformed with plasmids containing either the full-length hph gene (pPHT1, pMhph004, and pMhphi004) or the truncated hph gene (pAN7-1, phph004, and phphi004) in the presence of PEG-CaCl 2 were screened for the hygromycin-resistant colonies on PDAS plates overlaid with hygromycin.…”

Section: Resultsmentioning

confidence: 99%

“…Many wild isolates of basidiomycetes are dikaryotic and this also reduces the utility of such a method, given the likely difficulty of disrupting the desired target in both nuclear types. RNAi-mediated gene silencing is an alternative mechanism to targeted gene disruption and has already been established in species such as C. cinerea, A. bisporus, and S. commune, with the advantage that targeted insertion is not necessary and that it is likely to deplete specific transcripts irrespective of nuclear origin (8,9,18,37,56) so it would be phenotypically dominant.…”

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confidence: 99%

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Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianus

Kilaru

Collins

Hartley

et al. 2009

Appl Environ Microbiol

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We describe efficient polyethylene glycol (PEG)-mediated and Agrobacterium-mediated transformation systems for a pharmaceutically important basidiomycete fungus, Clitopilus passeckerianus, which produces pleuromutilin, a diterpene antibiotic. Three dominant selectable marker systems based on hygromycin, phleomycin, and carboxin selection were used to study the feasibility of PEG-mediated transformation of C. passeckerianus. The PEG-mediated transformation of C. passeckerianus protoplasts was successful and generated hygromycinresistant transformants more efficiently than either phleomycin or carboxin resistance. Agrobacterium-mediated transformation with plasmid pBGgHg containing hph gene under the control of the Agaricus bisporus gpdII promoter led to hygromycin-resistant colonies and was successful when homogenized mycelium and fruiting body gill tissue were used as starting material. Southern blot analysis of transformants revealed the apparently random integration of the transforming DNA to be predominantly multiple copies for the PEG-mediated system and a single copy for the Agrobacterium-mediated system within the genome. C. passeckerianus actin and tubulin promoters were amplified from genomic DNA and proved successful in driving green fluorescent protein and DsRed expression in C. passeckerianus, but only when constructs contained a 5 intron, demonstrating that the presence of an intron is prerequisite for efficient transgene expression. The feasibility of RNA interference-mediated gene silencing was investigated using gfp as a target gene easily scored in C. passeckerianus. Upon transformation of gfp antisense constructs into a highly fluorescent strain, transformants were recovered that exhibited either reduced or undetectable fluorescence. This was confirmed by Northern blotting showing depletion of the target mRNA levels. This demonstrated that gene silencing is a suitable tool for modulating gene expression in C. passeckerianus. The molecular tools developed in this study should facilitate studies aimed at gene isolation or characterization in this pharmaceutically important species.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianus

Kilaru

Collins

Hartley

et al. 2009

Appl Environ Microbiol

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“…Nevertheless, these data clearly demonstrated that RNA silencing can be efficiently induced by direct physical delivery of dsRNA into the cell (Caribé dos Santos et al, 2009). Similarly, in Coprinopsis cinerea the commercially available pSUPER RNAi System™ (OligoEngine™, USA) was tested (Costa et al, 2008). This system is based on a short hpRNA construct comprising a user defined sense and antisense target sequence separated by a 9 nt spacer.…”

Section: Rna Silencing As Defense Against Invasive Nucleic Acidsmentioning

confidence: 99%

“…The efficacy of a 19 nt stem length construct at inducing gfp silencing was investigated. Not unexpectedly, the efficiency of silencing was found to vary, with one strain showing an almost complete loss of gfp expression (Costa et al, 2008).…”

Section: Rna Silencing As Defense Against Invasive Nucleic Acidsmentioning

confidence: 99%

RNA silencing in fungi

et al. 2010

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RNA silencing is an evolutionarily conserved mechanism in eukaryotic organisms induced by doublestranded RNA (dsRNA) and plays an essential role in regulating gene expression and maintaining genome stability. RNA silencing occurs at both posttranscriptional levels through sequence-specific RNA degradation or translational repression and at transcriptional levels through RNA-directed DNA methylation and/or heterochromatin formation. RNA silencing pathways have been relatively well characterized in plants and animals, and are now also being widely investigated in diverse fungi, some of which are important plant pathogens. This review focuses primarily on the current understanding of the dsRNA-mediated posttranscriptional gene silencing processes in fungi, but also discusses briefly the known gene silencing pathways that appear to be independent of the RNA silencing machineries. We review RNA silencing studies for a variety of fungi and highlight some of the mechanistic differences observed in different fungal organisms. As RNA silencing is being exploited as a technology in gene function studies in fungi as well as in engineering anti-fungal resistance in plants and animals, we also discuss the recent progress towards understanding dsRNA uptake in fungi.

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“…RT-qPCR analyses indicated that transcripts of two Agaricus bisporus genes (URA3 and CBX) silenced with hairpin constructs were significantly reduced (Costa et al, 2009). In Coprinopsis cinerea, Costa et al (2008) reported that the use of short hairpin constructs promoted the formation of transformants with reduced transcripts of the Green Fluorescent Protein (GFP) transgene.…”

Section: Introductionmentioning

confidence: 99%

dsRNA-induced gene silencing in Moniliophthora perniciosa, the causal agent of witches’ broom disease of cacao

Santos

Sena

Santos

et al. 2009

Fungal Genetics and Biology

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The genome sequence of the hemibiotrophic fungus Moniliophthora perniciosa revealed genes possibly participating in the RNAi machinery. Therefore, studies were performed in order to investigate the efficiency of gene silencing by dsRNA. We showed that the reporter gfp gene stably introduced into the fungus genome can be silenced by transfection of in vitro synthesized gfpdsRNA. In addition, successful dsRNA-induced silencing of endogenous genes coding for hydrophobins and a peroxiredoxin were also achieved. All genes showed a silencing efficiency ranging from 18% to 98% when compared to controls even 28d after dsRNA treatment, suggesting systemic silencing. Reduction of GFP fluorescence, peroxidase activity levels and survival responses to H(2)O(2) were consistent with the reduction of GFP and peroxidase mRNA levels, respectively. dsRNA transformation of M. perniciosa is shown here to efficiently promote genetic knockdown and can thus be used to assess gene function in this pathogen.

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Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes

Cited by 18 publications

References 19 publications

Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianus

Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianus

RNA silencing in fungi

dsRNA-induced gene silencing in Moniliophthora perniciosa, the causal agent of witches’ broom disease of cacao

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