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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.

DOI: 10.7287/peerj.preprints.3177v1/supp-1

Additional Information

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Shelby S Calkins performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Nicole C Elledge performed the experiments, reviewed drafts of the paper.

Stephen M. Marek reviewed drafts of the paper.

M B. Couger performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, reviewed drafts of the paper.

Mostafa S Elshahed analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Noha H Youssef conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

DNA Deposition

The following information was supplied regarding the deposition of DNA sequences:

This Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GFSU00000000 (https://www.ncbi.nlm.nih.gov/nuccore/GFSU00000000). The version described in this paper is the first version, GFSU01000000 (https://www.ncbi.nlm.nih.gov/nuccore/GFSU01000000).

Data Deposition

The following information was supplied regarding data availability:

The research in this article did not generate any data or code (please explain).

Funding

This work was supported by the National Science Foundation Grant award number 1557102 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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