siRNA as a molecular tool for use in Aspergillus niger

Barnes, S.; Alcocer, Marcos; Archer, David B.

doi:10.1007/s10529-007-9614-0

Cited by 20 publications

(17 citation statements)

References 23 publications

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“…Therefore, it would seem reasonable that higher concentrations would be required to be effective using this approach in filamentous fungi. The concentrations used in this study are similar to those reported for Aspergillus (21,28). At the highest level, we used 100 nM (each) pooled mus53-directed siRNAs and observed a 65% reduction in mus53 mRNA in protoplasts.…”

Section: Discussionmentioning

confidence: 56%

“…The mus53 expression is relative to that with control treatment. 21 or 36 h, there was little statistical difference between using GS lipid and not using GS lipid.…”

Section: Figmentioning

confidence: 84%

“…The siRNA treatment method for lowering mRNA levels of specific target genes is simple and fully compatible with the standard T. reesei protoplast transformation method. In studies performed in A. niger, siRNAs were delivered to protoplasts and recovered on a solid matrix in a standard transformation method to reduce the expression of a heterologous Escherichia coli ␤-glucuronidase gene (21). Other investigators performed a simple treatment of Aspergillus nidulans germinating spores with naked siRNAs to silence polyamine biosynthesis genes and examined the effects on germ tube formation (22).…”

mentioning

confidence: 99%

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Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei

Chum

Schmidt

Saloheimo

et al. 2017

Appl Environ Microbiol

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Trichoderma reesei is a filamentous fungus that is used worldwide to produce industrial enzymes. Industrial strains have traditionally been created though systematic strain improvement using mutagenesis and screening approaches. It is also desirable to specifically manipulate the genes of the organism to further improve and to modify the strain. Targeted integration in filamentous fungi is typically hampered by very low frequencies of homologous recombination. To address this limitation, we have developed a simple transient method for silencing genes in T. reesei. Using gene-specific small interfering RNAs (siRNAs) targeted to mus53, we could achieve up to 90% knockdown of mus53 mRNA. As a practical example, we demonstrated that transient silencing of DNA repair genes significantly improved homologous integration of DNA at a specific locus in a standard protoplast transformation. The best transient silencing of mus53 with siRNAs in protoplasts could achieve up to 59% marker gene integration. IMPORTANCEThe previous solution for improving targeted integration efficiency has been deleting nonhomologous end joining (NHEJ) DNA repair genes. However, deleting these important repair genes may lead to unintended consequences for genomic stability and could lead to the accumulation of spontaneous mutations. Our method of transiently silencing NHEJ repair pathway genes allows recovery of their important repair functions. Here we report a silencing approach for improving targeted DNA integration in filamentous fungi. Furthermore, our transient silencing method is a truly flexible approach that is capable of knocking down the expression of a target gene in growing mycelial cultures, which could facilitate the broad study of gene functions in T. reesei.KEYWORDS Trichoderma reesei, filamentous fungi, gene silencing, industrial biotechnology, siRNA T richoderma reesei is a filamentous fungus that is used worldwide as a host for industrial enzyme production. The enzymes produced are used, for example, in pulp and paper production (1), in the food and feed industries (2), and in the textile industry (3, 4). T. reesei enzymes are also increasingly important because of their ability to turn lignocellulosic biomass into sugars that can be used to produce biofuels and chemicals (5, 6). The hydrolyzing enzymes (cellulase and hemicellulase) that are used to degrade lignocellulosic polysaccharides into fermentable sugars contribute substantially to the cost of bioethanol production (7). Systematic strain improvement through mutagenesis and screening has given rise to industrial strains producing over 100 g/liter extracellular proteins, with around one-half of the secreted proteins being the main cellulase, cellobiohydrolase I (8). Further improvements must be made, however, to make the production even more cost-effective and to improve the performance of the produced enzymes. Specific genetic manipulation of the enzyme production organism is a

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Section: Discussionmentioning

confidence: 56%

“…The mus53 expression is relative to that with control treatment. 21 or 36 h, there was little statistical difference between using GS lipid and not using GS lipid.…”

Section: Figmentioning

confidence: 84%

mentioning

confidence: 99%

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Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei

Chum

Schmidt

Saloheimo

et al. 2017

Appl Environ Microbiol

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“…To investigate whether this RNA uptake mechanism can be exploited to induce gene silencing in fungi, direct delivery of synthetic dsRNA or siRNA into fungal protoplasts or germinating spores has been examined in many studies. In A. fumigatus and A. nidulans , introduction of 21 and 23 nt-siRNAs into germinating spores induced sequence-specifi c gene silencing at least 72 h after treatment (Jochl et al 2009 ;Khatri and Rajam 2007 ;Barnes et al 2008 ).…”

Section: Direct Dsrna or Sirna Delivery Into Fungal Cellsmentioning

confidence: 99%

RNA Silencing in Filamentous Fungi: From Basics to Applications

Quốc

Nakayashiki

2014

Fungal Biology

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“…RNA interference (RNAi), mediated by small interfering RNA (siRNA), can induce post-transcriptional gene silencing in a highly potent and sequence-specific manner (Barnes et al 2008). However, synthetic siRNA or most vector-based siRNA demonstrated only transient knockdown of gene expression.…”

Section: Introductionmentioning

confidence: 99%

Locally administered lentivirus-mediated siRNA inhibits wear debris-induced inflammation in murine air pouch model

2008

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Lentivirus(LV)-mediated small interfering RNA (siRNA) targeting tumor necrosis factor-alpha (TNF-alpha) was locally administered into the air pouch of mice to inhibit inflammation induced by titanium alloy particles. The lentiviral vector expressed green fluorescent protein (GFP) as a reporter gene. Down-regulation of TNF-alpha in pouch area was confirmed by real-time PCR and ELISA, resulting in significantly decreased local inflammatory responses (P < 0.01). This approach was proven safe by localized GFP fluorescence and invariant TNF-alpha expression in peripheral blood, liver, spleen, kidney, lung and brain of mouse. In conclusion, locally administered siRNA provides an effective and safe method for inhibiting particle-induced inflammation.

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siRNA as a molecular tool for use in Aspergillus niger

Cited by 20 publications

References 23 publications

Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei

Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei

RNA Silencing in Filamentous Fungi: From Basics to Applications

Locally administered lentivirus-mediated siRNA inhibits wear debris-induced inflammation in murine air pouch model

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