Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungus Pecoramyces ruminantium strain C1A
- Published
- Accepted
- Subject Areas
- Molecular Biology, Mycology
- Keywords
- RNA interference, Pecoramyces ruminantium, D-lactate dehydrogenase, anaerobic gut fungi
- Copyright
- © 2017 Calkins et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2017. Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungus Pecoramyces ruminantium strain C1A. PeerJ Preprints 5:e3177v1 https://doi.org/10.7287/peerj.preprints.3177v1
Abstract
Members of the anaerobic gut fungi (AGF) reside in rumen, hindgut, and feces of ruminant and non-ruminant herbivorous mammals and reptilian herbivores. No protocols for gene insertion, deletion, silencing, or mutation are currently available for the AGF, rendering gene-targeted molecular biological manipulations unfeasible. Here, we developed and optimized an RNA interference (RNAi)-based protocol for targeted gene silencing in the anaerobic gut fungus Pecoramyces ruminantium strain C1A. Analysis of the C1A genome identified genes encoding enzymes required for RNA silencing in fungi (Dicer, Argonaute, Neurospora crassa QDE-3 homolog DNA helicase, Argonaute-interacting protein, and Neurospora crassa QIP homolog exonuclease); and the competency of C1A germinating spores for RNA uptake was confirmed using fluorescently labeled small interfering RNAs (siRNA). Addition of chemically-synthesized siRNAs targeting D-lactate dehydrogenase (ldhD) gene to C1A germinating spores resulted in marked target gene silencing; as evident by significantly lower ldhD transcriptional levels, a marked reduction in the D-LDH specific enzymatic activity in intracellular protein extracts, and a reduction in D-lactate levels accumulating in the culture supernatant. Comparative transcriptomic analysis of untreated versus siRNA-treated cultures identified a few off-target siRNA-mediated gene silencing effects. As well, significant differential up-regulation of the gene encoding NAD-dependent 2-hydroxyacid dehydrogenase (Pfam00389) in siRNA-treated C1A cultures was observed, which could possibly compensate for loss of D-LDH as an electron sink mechanism in C1A. The results demonstrate the feasibility of RNAi in anaerobic fungi, and opens the door for gene silencing-based studies in this fungal clade.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
Supplementary material for : Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungus Pecoramyces ruminantium strain C1A
Figure S1. Uptake of fluorescently (Cy3) tagged siRNA by C1A spores. (A) The ldhD-specific siRNA was added to the flooding solution 75 minutes after the onset of flooding followed by incubation for 15 more minutes at 39ºC. Samples (a few microliters) were taken at regular intervals for visualization. The same field is shown for DAPI-, and Cy3-labeled germinating spores (Note that the spores were concurrently stained with DAPI and fluorescing green indicating the uptake of the Cy3-labeled siRNA) (bar=20 μm). (B) Effect of the siRNA treatment on fungal growth rate. siRNA-treated spores were collected and used to inoculate fresh RFC medium. Control cultures were started at the same time using siRNA-untreated spores. Headspace pressure was measured daily and used to calculate fungal biomass as described previously (1). Error bars are standard deviations from at least three replicate cultures for each condition.
Table S1. Transcripts with a significant (False Discovery Rate (FDR) < 0.1) fold change in the ldhD-siRNA-treated cultures.