A copper-controlled RNA interference system for reversible silencing of target genes in Trichoderma reesei

Xiangfeng Meng,Lei Wang,Guanjun Chen,Fanglin Zheng,Weixin Zhang,Weifeng Liu,Yaohua Zhong

doi:10.1186/s13068-018-1038-7

Abstract

BackgroundTrichoderma reesei is a primary lignocellulosic enzyme producer in industry. However, the mechanisms underlying cellulase synthesis as well as other physiological processes are insufficiently understood partly due to the sophisticated process for its genetic manipulation. Target gene knockdown by RNA interference (RNAi) is a powerful tool for genetic research and biotechnology in eukaryotes including filamentous fungi. Previously reported RNAi system in T. reesei was either uncontrollable or only applicable in certain nutrition state.ResultsIn the present study, we incorporated the copper-responsive tcu1 promoter into an RNAi-mediated silencing system to develop a controllable RNAi-mediated silencing system in T. reesei. As the proof-of-concept, a prototrophic pyr4 gene, highly expressed cel7a and xyr1 genes induced by Avicel and a fab1 gene, whose knockout has proved to be intractable, were successfully knocked down in the absence of copper when the respective RNAi fragment was expressed. Importantly, the phenotype of RNAi strains was shown to be reversed easily to mimic the complementation for excluding any unwanted effects resulted from the random integration of the hpRNA cassette by adding copper in the media. Thus, this controllable RNAi-mediated silencing system can be turned on and turned off only depending on the absence and presence of copper ions in the media, respectively, and not on the nutritional states.ConclusionsThe copper-controlled RNA interference system represents an effective tool for reversible silencing of target genes in T. reesei. This reported strategy to conditionally knock down or turn off genes will contribute to our understanding of T. reesei gene functions, especially those that are difficult to be knocked out due to various reasons. In addition, this simple and cost-effective method holds great potential for the application in synthetic biology and genetic engineering of T. reesei.

Highlights

Trichoderma reesei is a primary lignocellulosic enzyme producer in industry
Design of copper‐responsive RNA interference (RNAi)‐mediated silencing system The controllability of the Ptcu1 promoter was incorporated into an RNAi-mediated silencing system in T. reesei
The transcription of hairpin RNA (hpRNA) fragments for target genes will result in the formation of double-stranded RNA that is recognized by Dicer and produces small interfering RNA (siRNA) to induce degradation of target mRNAs

Summary

Introduction

Trichoderma reesei is a primary lignocellulosic enzyme producer in industry. The large amounts of lignocellulosic enzymes (e.g., cellulases and hemicellulases) secreted by the ascomycete Trichoderma reesei represent the prominent source of enzyme cocktails that are used in industry for biomass degradation [10,11,12]. Industrial T. reesei strains have been reported to produce cellulases over 100 g/L [13], the cost of enzyme production used in the saccharification step is still considered to be the major bottleneck of biomass conversion for the economical production of biofuels and biochemicals. The production of lignocellulosic enzymes by T. reesei involves many physiological processes including extracellular signal sensing [14, 15], intracellular signal transduction [16], transcriptional regulation of cellulase genes and protein synthesis [17, 18], and secretion [19]. Understanding the molecular mechanism behind these processes is crucial to further improve cellulase productivity of T. reesei

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