Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungus <i>Pecoramyces ruminantium</i> strain C1A

Calkins, Shelby; Elledge, Nicole C.; Marek, Stephen M.; Couger, Matthew Brian; Elshahed, Mostafa S.; Youssef, Noha H.

doi:10.7287/peerj.preprints.3177v1

Cited by 2 publications

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“…Isolation of AGF taxa enables taxonomic, metabolic, physiological and ultrastructural characterization of individual taxa. As well, cultures availability enables subsequent—omics, synthetic and system‐biology, and biogeography‐based investigations (Couger et al ., 2015; Seppälä et al ., 2016; Haitjema et al ., 2017; Calkins et al ., 2018; Henske et al ., 2018a; Henske et al ., 2018b; Murphy et al ., 2019), as well as evaluation of evolutionary processes underpinning speciation in the AGF (Youssef et al ., 2013; Wang et al ., 2019). However, efforts to isolate and maintain AGF strains have lagged behind their aerobic counterparts mainly due to their strict anaerobic nature and the lack of reliable long‐term storage procedures.…”

Section: Introductionmentioning

confidence: 99%

Assessing anaerobic gut fungal diversity in herbivores using D1/D2 large ribosomal subunit sequencing and multi‐year isolation

Hanafy

Johnson

Youssef

2020

Environmental Microbiology

111

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The anaerobic gut fungi (AGF, Neocallimastigomycota) reside in the alimentary tracts of herbivores where they play a central role in the breakdown of plant material. Here, we report on the development of the hypervariable domains D1/D2 of the large ribosomal subunit (D1/D2 LSU) as a barcoding marker for the AGF. We generated a reference D1/D2 LSU database for all cultured AGF genera, as well as the majority of candidate genera encountered in prior internal transcribed spacer 1 (ITS1)-based surveys. Subsequently, a D1/D2 LSUbased diversity survey using long read PacBio SMRT sequencing was conducted on faecal samples from 21 wild and domesticated herbivores. Twenty-eight genera and candidate genera were identified, including multiple novel lineages that were predominantly, but not exclusively, identified in wild herbivores. Association between certain AGF genera and animal lifestyles, or animal host family was observed. Finally, to address the current paucity of AGF isolates, concurrent isolation efforts utilizing multiple approaches to maximize recovery yielded 216 isolates belonging to 12 different genera, several of which have no prior cultured-representatives. Our results establish the utility of D1/D2 LSU and PacBio sequencing for AGF diversity surveys, the culturability of multiple AGF taxa, and demonstrate that wild herbivores represent a yet-untapped reservoir of AGF diversity.

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Section: Introductionmentioning

confidence: 99%

Assessing anaerobic gut fungal diversity in herbivores using D1/D2 large ribosomal subunit sequencing and multi‐year isolation

Hanafy

Johnson

Youssef

2020

Environmental Microbiology

111

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“…More recently, however, chemically synthesized dsRNA has been used for interference targeting the RISC complex genes in many fungal species [19,20]. The direct uptake of small RNAs by fungal cells for gene knockdown has been reported in monokaryons, for example, in germinating spores [21,22]. Bioinformatic tools have been developed in RNAi design to predict the efficiency of the silencing and to estimate the probability of off-targets [23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Geosmin synthase ges1 knock‐down by siRNA in the dikaryotic fungus Tricholoma vaccinum

et al. 2021

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Genetic manipulation for generating knock-out experiments is essential in deciphering the precise function of a gene. However, dikaryotic fungi pose the inherent challenge of having two allelic versions of each gene, one in each nucleus. In addition, they often are slow-growing and do not withstand protoplasting, which is why Agrobacterium tumefaciens-mediated transformation has been adapted. To obtain knock-out strains, however, is not feasible with a mere deletion construct transformation and screening for deletions in both nuclear copies. Hence, a convenient method using chemically synthesized dicer substrate interfering RNA (DsiRNA) for posttranscriptional interference of targeted mRNA was developed, based on the fungal dicer/argonaute system inherent in fungi for sequence recognition and degradation. A proof-ofprinciple using this newly established method for knock-down of the volatile geosmin is presented in the dikaryotic fungus Tricholoma vaccinum that is forming ectomycorrhizal symbiosis with spruce trees. The gene ges1, a terpene synthase, was transcribed with a 50-fold reduction in transcript levels in the knockdown strain. The volatile geosmin was slightly reduced, but not absent in the fungus carrying the knockdown construct pointing at low specificity in other terpene synthases known for that class of enzymes.

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Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungus Pecoramyces ruminantium strain C1A

Cited by 2 publications

References 0 publications

Assessing anaerobic gut fungal diversity in herbivores using D1/D2 large ribosomal subunit sequencing and multi‐year isolation

Assessing anaerobic gut fungal diversity in herbivores using D1/D2 large ribosomal subunit sequencing and multi‐year isolation

Geosmin synthase ges1 knock‐down by siRNA in the dikaryotic fungus Tricholoma vaccinum

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