T7-RNA polymerase dependent RNAi system inAspergillus fumigatus: a proof of concept study

Abstract

An RNAi system based on T7 RNA polymerase (TRNAP) was designed and examined in Aspergillus fumigatus. This system consists of two elements; an inducible T7RNAP expressing cassette and an AMA1-based episomal RNAi plasmid. These constructs were transformed into the A. fumigatus protoplasts and the efficiency of this system was tested in downregulation of alb1 gene. Upon the induction of T7RNAP expression, the recombinant T7RNAP was able to recognize T7 promoters, which were located on the episomal plasmid and in opposite direction. As a result, the bidirectional transcription of alb1 fragment led to the silencing of the target gene. However, our results demonstrated that this silencing system is unstable and may not be applicable in preparation of RNAi libraries.

Silencing Dicer-Like Genes Reduces Virulence and sRNA Generation in Penicillium italicum, the Cause of Citrus Blue Mold

“…This approach includes a key step of incubating dsRNA with citrus wounds, which achieved a gene silencing efficiency similar to that of incubating dsRNA with P. italicum protoplasts (Figure 4); doing this incubation before the infection is also a key step in protoplast-mediated RNAi. Indeed, protoplast-mediated RNAi without the barrier of the fungal cell wall has proven to be immensely successful in filamentous fungi [34,35]. It is accepted that exosomes (extracellular vesicles) play a central role in cell-to-cell communication, for example, Arabidopsis secretes exosome-like extracellular vesicles to deliver sRNA into Botrytis cinerea to silence virulence-related genes [7], and exosomes are involved in the transfer of small RNAs by nematode parasites to mammalian cells [36].…”

“…A total of 600-700 sRNAs were obtained from Trichoderma reesei by Solexa sequencing, but only 13 milRNAs were predicted based on hairpin structure analysis [49]. Indeed, dsRNA/siRNA from a vector expressing the transgene construct also worked well, with specific efficiency to silence related genes in fungi [34,35]. Consequently, we need to develop a pipeline to identify siRNA production and a reliable methodology to experimentally characterize functions of siRNAs in fungal species in the future.…”

Development of Host‐Orthogonal Genetic Systems for Synthetic Biology

“…[ 86 ] Recently, a T7 RNAP‐based RNA interference (RNAi) system was designed and detected in Aspergillus fumigatus , an important airborne fungal pathogen. [ 87 ] The system consists of two elements: an inducible T7 RNAP expressing cassette and an apical membrane antigen (AMA1)‐based episomal RNAi plasmid. However, this silencing system is unstable and may not be applicable in preparation of RNAi libraries.…”

RNA Interference as a Prospective Tool for the Control of Human Viral Infections

“…A possibility to generate dsRNA in mammalian or bacterial cells, using viral RNA polymerases, has been demonstrated, which potentially allows to scale-up the production at low cost (Aalto et al, 2007 ; Heninger and Buchholz, 2007 ; Huang and Lieberman, 2013 ; Enayati et al, 2016 ). However, these systems still need further elaboration and most probably will not have clinical applications but, for instance, can be used in agriculture (Tenllado et al, 2003 ; Niehl et al, 2018 ).…”