milR4 and milR16 Mediated Fruiting Body Development in the Medicinal Fungus Cordyceps militaris

Abstract

Cordyceps militaris readily performs sexual reproduction, thus providing a remarkably rich model for understanding the processes involved in sexual development. It could regulate expression of human genes by diet-derived miRNA-like RNAs (milRNAs). However, the study of miRNAs in C. militaris has been limited. In the present study, genes encoding Dicers, Argonautes, and RNA-dependent RNA polymerases were identified. Illumina deep sequencing was performed to characterize the milRNAs in C. militaris at asexual and sexual development stages. Total 38 milRNAs were identified and five milRNAs were validated by northern blot and qRT-PCR, out of which, 19 were specific for sexual development. Importantly, the fungi could not form fruiting bodies after disruption of milR4, while the perithecium was formed in advance after over-expression of milR4. Abnormal pale yellow fruiting body primordium, covered with abnormal primordium, was formed in the strain with miR16 disruption. Although no milR4 or milR16 target genes were identified, differential expression of many different genes involved in mycelium growth and sexual development (mating process, mating signaling, and fruiting body development) among these mutants were found. Overall, milRNAs play vital roles in sexual development in C. militaris.

Unmatched Level of Molecular Convergence among Deeply Divergent Complex Multicellular Fungi

“…Argonaute-family proteins were developmentally regulated in all nine species, suggesting RNA interference as a widespread regulatory mechanism of CM morphogenesis, consistent with recent reports on individual species ( Lau et al. 2018 ; Shao et al. 2019 ).…”

The Genome of the Medicinal Macrofungus Sanghuang Provides Insights Into the Synthesis of Diverse Secondary Metabolites

“…Total RNA was extracted at different asexual developmental stages of S. sanghuang, i.e., 10 days old (M10d) and 20 days old (M20d), as well as from different sexual developmental stages, i.e., one year old (OY) and three year old (TY). The cDNA was synthesized and quantitative PCR was performed as reported previously (Shao et al, 2019). qPCR amplification was performed with a SYBR Green kit (Takara) and Bio-Rad CFX96TM system (Hercules, CA, United States).…”

mRNA-seq and miRNA-seq Profiling Analyses Reveal Molecular Mechanisms Regulating Induction of Fruiting Bodies in Ophiocordyceps Sinensis

“…With the development of high-throughput sequencing technology, the existence of miRNA or milRNAs in O. sinensis have already been shown. Here, 106 novel milRNAs were identi ed from our sRNA-Seq data, which greatly enrich the miRNA database of this Cordyceps fungi 16,18 . miRNAs regulate gene expression mainly by promoting cleavage of the target mRNAs or by regulating TFs; therefore, DEM-DEG target gene relationship pairs can re ect the regulatory function of milRNAs more accurately.…”

“…The small RNA-mediated RNA interference mechanism plays an important role in the ne-tuning of the transcriptome during ascospore formation in Fusarium graminearum 17 . In Cordyceps militaris, disruption and overexpression of candidates milR4 and milR16 con rmed that milRNAs in C. militaris regulate fruiting body formation 18 . These studies suggested that milRNAs may play important roles in the regulation of development in O. sinensis.…”