RNAi-mediated gene silencing of highly expressed genes in the industrial fungi Trichoderma reesei and Aspergillus niger

Abstract

RNA interference utilizes small interfering RNAs of 20–25 nucleotides processed from double-stranded RNA to initiate sequence-specific digestion of the cognate messenger RNA. We sought to investigate whether use of RNAi could be extended to industrially important filamentous fungi by determining if suppression of highly expressed fungal genes can be achieved and then maintained during growth in liquid media. Aspergillus niger and Trichoderma reesei are capable of secreting abundant amounts of amyloglucosidase and cellobiohydrolase II (CEL6A), respectively. A vector directing expression of a T. reesei cel6a hairpin RNA was used to transform T. reesei strain RutC30. Greater than 50% of transformants displayed some measure of CEL6A suppression, with a portion of these transformants appearing to be fully suppressed. Steady-state levels of cel6a-specific mRNA in these transformants, determined by Northern blots, paralleled extracellular yields of CEL6A visualized by SDS-PAGE. Decreased steady-state levels of cel6A-specific mRNA were confirmed using quantitative RT-PCR. In addition, cel6a-specific siRNAs were identified in transformants exhibiting decreased levels of cel6a mRNA and CEL6A protein, confirming RNAi as the process responsible for achieving gene silencing. Furthermore, RNAi-mediated gene silencing was maintained throughout both a seven-day fermentation and continuous growth over 35 days in shake flasks. Efficient gene silencing was also accomplished in A. niger, where apparently complete suppression of the amyloglucosidase (glaA) gene was achieved. These results demonstrate the robustness and stability of RNAi in T. reesei and A. niger and support the utility of this technology for strain development programs and functional genomics studies in these and possibly other industrial fungal strains.