A fast and reliable procedure for spore collection from anaerobic fungi: Application for RNA uptake and long-term storage of isolates

Abstract

Anaerobic gut fungi (AGF) represent a basal fungal lineage (phylum Neocallimastigomycota) that resides in the rumen and alimentary tracts of herbivores. The AGF reproduce asexually, with a life cycle that involves flagellated zoospores released from zoosporangia followed by encystment, germination and the subsequent development of rhizomycelia. A fast and reliable approach for AGF spore collection is critical not only for developmental biology studies, but also for molecular biological (e.g. AMT-transformation and RNAi) approaches. Here, we developed and optimized a simple and reliable procedure for the collection of viable, competent, and developmentally synchronized AGF spores under strict anaerobic conditions. The approach involves growing AGF on agar medium in serum bottles under anaerobic conditions, and flooding the observed aerial growth to promote spore release from sporangia into the flooding suspension. The released spores are gently collected using a wide bore sterile needle. Process optimization resulted in the recovery of up to 7×10(9) spores per serum bottle. Further, the released spores exhibited synchronized development from flagellated spores to encysted spores and finally to germinating spores within 90min from the onset of flooding. At the germinating spore stage, the obtained spores were competent, and readily uptook small interfering RNA (siRNA) oligonucleotides. Finally, using multiple monocentric and polycentric AGF isolates, we demonstrate that AGF grown on agar surface could retain viability for up to 16weeks at 39°C, and hence this solid surface growth procedure represents a simple, cryopreservative- and freezing temperature-free approach for AGF storage.

Horizontal Gene Transfer as an Indispensable Driver for Evolution of Neocallimastigomycota into a Distinct Gut-Dwelling Fungal Lineage

“…According to the weak-link model (68), these weakly protected and exposed structures provide excellent entry point of foreign DNA to eukaryotic genomes. It is important to note that AGF zoospores also appear to be naturally competent, capable of readily taking up nucleic acids from their surrounding environment (69).…”

“…Microscopic examination of thallus growth pattern, rhizoid morphology, and zoospore flagellation, as well LSU rRNA gene D1-D2 domain amplification and sequencing, was employed to determine the genus-level affiliation of all isolates (74). Isolates were maintained and routinely subcultured on rumen fluid medium supplemented with antibiotics (to guard against accidental bacterial contamination) and stored on agar media as described previously (42,69).…”

Seven new Neocallimastigomycota genera from fecal samples of wild, zoo-housed, and domesticated herbivores: Description ofGhazallomyces constrictusgen. nov., sp. nov.,Aklioshbomyces papillarumgen. nov., sp. nov.,Agriosomyces longusgen. nov., sp. nov.,Capellomyces foraminisgen. nov., sp. nov. andCapellomyces elongatussp. nov.,Joblinomyces apicalisgen. nov., sp. nov.,Khoyollomyces ramosusgen. nov., sp. nov., andTahromyces munnarensisgen. nov., sp. nov

“…Long-term storage was conducted by surface inoculation of RF-cellobiose agar media as described previously (Calkins and others 2016), or by cryopreservation at −80 °C using 0.64 M ethylene glycol as the cryoprotectant (Callaghan and others 2015). Cultures are available at Oklahoma State University, Department of Microbiology and Molecular Genetics culture collection, and at MACS Collection of Microorganisms (MCM), Agharkar Research Institute, Pune, India.…”

“…In the USA, isolation of anaerobic fungal strains was conducted as previously described (Hanafy and others 2017). Feces were suspended in rumen-fluid (RF) media (Calkins and others 2016; Hanafy and others 2018) with either cellobiose, or 0.5% cellobiose and switchgrass (0.5%) used as a substrate. Antibiotics (50 μg/mL kanamycin, 50 μg/mL penicillin, 20 μg/mL streptomycin, and 50 μg/mL chloramphenicol, respectively) were added to inhibit growth of bacteria.…”

Peer Review #1 of "Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungus Pecoramyces ruminantium strain C1A (v0.1)"

“…Microorganism and culture maintenance. Pecoramyces ruminantium strain C1A was isolated previously in our laboratory (56) and maintained by biweebly transfers into an antibioticsupplemented rumen-fluid-cellobiose medium (RFC) as described previously (57).…”