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.
Highlights
MicroRNAs are small regulatory RNA molecules (18–24 nt) that play a pervasive role in gene regulation and influence a variety of biological processes in animals, plants, viruses, and fungi (Reis, 2017; Zeng et al, 2018)
Genome of C. militaris2 was searched and two RNA-dependent RNA polymerase (RDRP), two AGOs, and two Dicers were identified. To annotate of these genes, Phylogeny trees were generated with RDRP, AGO, and Dicer genes from other eight fungal species, including the entomopathogenic fungi (M. anisopliae, Beauveria bassiana, O. sinensis), plant pathogenic fungi (Magnaporthe oryzae, F. graminearum, Cryphonectria parasitica), the model filamentous fungus N. crassa, a saprophytic filamentous fungus A. flavus
CMDicer-1, CMRDRP1, and CMAGO-3 were expressed with significant differences among different development stages, suggesting that CMDicer-1 and CMRDRP1 are the major genes that process dsRNA into mature miRNA-like RNAs (milRNAs), and CMAGO-3 is the major gene involved in the milRNA functioning in asexual and sexual development
Summary
MicroRNAs (miRNAs) are small regulatory RNA molecules (18–24 nt) that play a pervasive role in gene regulation and influence a variety of biological processes in animals, plants, viruses, and fungi (Reis, 2017; Zeng et al, 2018). To date, following the discovery of key components of miRNA maturation and function like RNA-dependent RNA polymerase (RDRP), argonaute (AGO), and Dicer proteins in the universal fungal species, hundreds of milRNAs have been identified from dozens of fungal species (Lee et al, 2010; Lau et al, 2013; Meng et al, 2017; Zeng et al, 2018). A recent report showed that C. militaris containing high levels of two milRNAs (milR1321 and milR3188) that target 3’-untranslated region of CXCR2 mRNA to inhibit its expression, alleviates severity of murine acute lung injury, suggesting the regulation of human gene expression by dietderived milRNAs in C. militaris, as reported previously (Liu et al, 2015). Only two milRNAs in C. militaris have been identified far, which inhibits the research for potential roles of diet-derived milRNAs on human gene expression
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