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Efficient cloning system for construction of gene silencing vectors in Aspergillus niger


An approach based on Gateway recombination technology to efficiently construct silencing vectors was developed for use in the biotechnologically important fungus Aspergillus niger. The transcription activator of xylanolytic and cellulolytic genes XlnR of A. niger was chosen as target for gene silencing. Silencing was based on the expression vector pXLNRir that was constructed and used in co-transformation. From all the strains isolated (N = 77), nine showed poor xylan-degrading activities in two semi-quantitative plate assays testing different activities for xylan degradation. Upon induction on d-xylose, transcript levels of xlnR were decreased in the xlnR-silenced strains, compared to a wild-type background. Under these conditions, the transcript levels of xyrA and xynB (two genes regulated by XlnR) were also decreased for these xlnR-silenced strains. These results indicate that the newly developed system for rapid generation of silencing vectors is an effective tool for A. niger, and this can be used to generate strains with a tailored spectrum of enzyme activities or product formation by silencing specific genes encoding, e.g., regulators such as XlnR.

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  1. Barnes SE, Alcocer MJC, Archer DB (2008) siRNA as a molecular tool for use in Aspergillus niger. Biotechnology Letters 30:885–890

  2. Benen JA, Kester HC, Visser J (1999) Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C. Eur J Biochem 259:577–585

  3. Berns K, Hijmans EM, Mullenders J, Brummelkamp TR, Velds A, Heimerikx M, Kerkhoven RM, Madiredjo M, Nijkamp W, Weigelt B, Agami R, Ge W, Cavet G, Linsley PS, Beijersbergen RL, Bernards R (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428:431–437

  4. Boutros M, Kiger AA, Armknecht S, Kerr K, Hild M, Koch B, Haas SA, Paro R, Perrimon N (2004) Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303:832–835

  5. Catalanotto C, Azzalin G, Macino G, Cogoni C (2000) Gene silencing in worms and fungi. Nature 404:245

  6. Catalanotto C, Pallotta M, ReFalo P, Sachs MS, Vayssie L, Macino G, Cogoni C (2004) Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa. Mol Cell Biol 24:2536–2545

  7. Cogoni C, Macino G (1997) Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa. Proc Natl Acad Sci U S A 94:10233–10238

  8. Cogoni C, Macino G (1999a) Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 399:166–169

  9. Cogoni C, Macino G (1999b) Posttranscriptional gene silencing in Neurospora by a RecQ DNA helicase. Science 286:2342–2344

  10. de Graaff L, van den Broek H, Visser J (1988) Isolation and transformation of the pyruvate kinase gene of Aspergillus nidulans. Curr Genet 13:315–321

  11. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811

  12. Fitzgerald A, Van Kan JA, Plummer KM (2004) Simultaneous silencing of multiple genes in the apple scab fungus, Venturia inaequalis, by expression of RNA with chimeric inverted repeats. Fungal Genet Biol 41:963–971

  13. Goldoni M, Azzalin G, Macino G, Cogoni C (2004) Efficient gene silencing by expression of double stranded RNA in Neurospora crassa. Fungal Genet Biol 41:1016–1024

  14. Goosen T, van Engelenburg F, Debets F, Swart K, Bos K, van den Broek H (1989) Tryptophan auxotrophic mutants in Aspergillus niger: inactivation of the trpC gene by cotransformation mutagenesis. Mol Gen Genet 219:282–288

  15. Hasper AA, Visser J, de Graaff LH (2000) The Aspergillus niger transcriptional activator XlnR, which is involved in the degradation of the polysaccharides xylan and cellulose, also regulates D-xylose reductase gene expression. Mol Microbiol 36:193–200

  16. Hasper AA, Trindade LM, van der Veen D, van Ooyen AJ, de Graaff LH (2004) Functional analysis of the transcriptional activator XlnR from Aspergillus niger. Microbiology 150:1367–1375

  17. Henry C, Mouyna I, Latge JP (2007) Testing the efficacy of RNA interference constructs in Aspergillus fumigatus. Curr Genet 51:277–284

  18. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, Welchman DP, Zipperlen P, Ahringer J (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421:231–237

  19. Karimi M, Inze D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195

  20. Kusters-van Someren MA, Harmsen JA, Kester HC, Visser J (1991) Structure of the Aspergillus niger pelA gene and its expression in Aspergillus niger and Aspergillus nidulans. Curr Genet 20:293–299

  21. Meyer V (2008) Genetic engineering of filamentous fungi—progress, obstacles and future trends. Biotechnol Adv 26:177–185

  22. Meyer V, Arentshorst M, El-Ghezal A, Drews AC, Kooistra R, van den Hondel CA, Ram AF (2007) Highly efficient gene targeting in the Aspergillus niger kusA mutant. J Biotechnol 128:770–775

  23. Moriwaki A, Ueno M, Arase S, Kihara J (2007) RNA-mediated gene silencing in the phytopathogenic fungus Bipolaris oryzae. FEMS Microbiology Lett 269:85–89

  24. Mouyna I, Henry C, Doering TL, Latge JP (2004) Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus. FEMS Microbiol Lett 237:317–324

  25. Nakayashiki H, Hanada S, Nguyen BQ, Kadotani N, Tosa Y, Mayama S (2005) RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet Biol 42:275–283

  26. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2:279–289

  27. Nicolas FE, Torres-Martinez S, Ruiz-Vazquez RM (2003) Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes. Embo J 22:3983–3991

  28. Paddison PJ, Silva JM, Conklin DS, Schlabach M, Li M, Aruleba S, Balija V, O’Shaughnessy A, Gnoj L, Scobie K, Chang K, Westbrook T, Cleary M, Sachidanandam R, McCombie WR, Elledge SJ, Hannon GJ (2004) A resource for large-scale RNA-interference-based screens in mammals. Nature 428:427–431

  29. Pel HJ, de Winde JH, Archer DB, Dyer PS, Hofmann G, Schaap PJ, Turner G, de Vries RP, Albang R, Albermann K, Andersen MR, Bendtsen JD, Benen JA, van den Berg M, Breestraat S, Caddick MX, Contreras R, Cornell M, Coutinho PM, Danchin EG, Debets AJ, Dekker P, van Dijck PW, van Dijk A, Dijkhuizen L, Driessen AJ, d’Enfert C, Geysens S, Goosen C, Groot GS, de Groot PW, Guillemette T, Henrissat B, Herweijer M, van den Hombergh JP, van den Hondel CA, van der Heijden RT, van der Kaaij RM, Klis FM, Kools HJ, Kubicek CP, van Kuyk PA, Lauber J, Lu X, van der Maarel MJ, Meulenberg R, Menke H, Mortimer MA, Nielsen J, Oliver SG, Olsthoorn M, Pal K, van Peij NN, Ram AF, Rinas U, Roubos JA, Sagt CM, Schmoll M, Sun J, Ussery D, Varga J, Vervecken W, van de Vondervoort PJ, Wedler H, Wosten HA, Zeng AP, van Ooyen AJ, Visser J, Stam H (2007) Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat Biotechnol 25:221–231

  30. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45

  31. Pontecorvo G, Roper JA, Hemmons LM, Macdonald KD, Bufton AW (1953) The genetics of Aspergillus nidulans. Adv Genet 5:141–238

  32. Romano N, Macino G (1992) Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6:3343–3353

  33. Ruijter GJ, Panneman H, Xu D, Visser J (2000) Properties of Aspergillus niger citrate synthase and effects of citA overexpression on citric acid production. FEMS Microbiol Lett 184:35–40

  34. Tanguay P, Bozza S, Breuil C (2006) Assessing RNAi frequency and efficiency in Ophiostoma floccosum and O. piceae. Fungal Genet Biol 43:804–812

  35. van der Krol AR, Mur LA, Beld M, Mol JN, Stuitje AR (1990) Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2:291–299

  36. van Peij NN, Gielkens MM, de Vries RP, Visser J, de Graaff LH (1998a) The transcriptional activator XlnR regulates both xylanolytic and endoglucanase gene expression in Aspergillus niger. Appl Environ Microbiol 64:3615–3619

  37. van Peij NN, Visser J, de Graaff LH (1998b) Isolation and analysis of xlnR, encoding a transcriptional activator co-ordinating xylanolytic expression in Aspergillus niger. Mol Microbiol 27:131–142

  38. Vishniac W, Santer M (1957) The thiobacilli. Bacteriol Rev 21:195–213

  39. Walti MA, Villalba C, Buser RM, Grunler A, Aebi M, Kunzler M (2006) Targeted gene silencing in the model mushroom Coprinopsis cinerea (Coprinus cinereus) by expression of homologous hairpin RNAs. Eukaryot Cell 5:732–744

  40. Yamada O, Ikeda R, Ohkita Y, Hayashi R, Sakamoto K, Akita O (2007) Gene silencing by RNA interference in the koji mold Aspergillus oryzae. Biosci Biotechnol Biochem 71:138–144

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This research was funded by the Kluyver Centre for Genomics of Industrial Fermentation, which is supported by the Netherlands Genomics Initiative.

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Correspondence to Leo H. de Graaff.

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Open Access This is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License (https://creativecommons.org/licenses/by-nc/2.0), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

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Oliveira, J.M., van der Veen, D., de Graaff, L.H. et al. Efficient cloning system for construction of gene silencing vectors in Aspergillus niger . Appl Microbiol Biotechnol 80, 917–924 (2008). https://doi.org/10.1007/s00253-008-1640-x

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  • pFIRD1
  • Gateway cloning
  • Aspergillus niger
  • Post-transcriptional gene silencing
  • XlnR