Dicer and Argonaute Genes Involved in RNA Interference in the Entomopathogenic Fungus Metarhizium robertsii

Abstract

RNA interference (RNAi) is a gene-silencing mechanism that plays an important role in gene regulation in a number of eukaryotic organisms. Two core components, Dicer and Argonaute, are central in the RNAi machinery. However, the physiological roles of Dicer and Argonaute in the entomopathogenic fungus Metarhizium robertsii have remained unclear. Here, the roles of genes encoding Dicer (M. robertsii dcl1 [Mrdcl1] and Mrdcl2) and Argonaute (Mrago1 and Mrago2) proteins in M. robertsii were investigated. The results showed that the Dicer-like protein MrDCL2 and Argonaute protein MrAGO1 are the major components of the RNAi process occurring in M. robertsii. The Dicer and Argonaute genes were not involved in the regulation of growth and diverse abiotic stress response in M. robertsii under the tested conditions. Moreover, our results showed that the Dicer and Argonaute gene mutants demonstrated reduced abilities to produce conidia, compared to the wild type (WT) and the gene-rescued mutant. In particular, the conidial yields in the Δdcl2 and Δago1 mutants were reduced by 55.8% and 59.3%, respectively, compared with those from the control strains. Subsequently, for the WT and Δdcl2 mutant strains, digital gene expression (DGE) profiling analysis of the stage of mycelium growth and conidiogenesis revealed that modest changes occur in development or metabolism processes, which may explain the reduction in conidiation in the Δdcl2 mutant. In addition, we further applied high-throughput sequencing technology to identify small RNAs (sRNAs) that are differentially expressed in the WT and the Δdcl2 mutant and found that 4 known microRNA-like small RNAs (milRNAs) and 8 novel milRNAs were Mrdcl2 dependent in M. robertsii. IMPORTANCEThe identification and characterization of components in RNAi have contributed significantly to our understanding of the mechanism and functions of RNAi in eukaryotes. Here, we found that Dicer and Argonaute genes play an important role in regulating conidiation in M. robertsii. Our study also demonstrates that diverse small RNA pathways exist in M. robertsii. The study provides a theoretical platform for exploration of the functions of Dicer and Argonaute genes involved in RNAi in fungi.KEYWORDS Argonaute, Dicer, Metarhizium robertsii, RNA interference R NA interference (RNAi) is a gene silencing mechanism that plays an important role in regulating gene expression from fungi, plants, and animals, and it has become a valuable molecular tool for analyzing the functions of many genes (1, 2). The 20-to 30-nucleotide (nt) small RNA (sRNA) molecules have emerged as powerful regulators of gene expression and genome stability via conserved eukaryotic RNAi-related pathways

Differential Functions of Two Metalloproteases, Mrmep1 and Mrmep2, in Growth, Sporulation, Cell Wall Integrity, and Virulence in the Filamentous Fungus Metarhizium robertsii

“…Phenotype assays were performed as described previously ( Meng et al, 2017 ). The conidial suspension (1 μL; concentration, 1 × 10 7 conidia mL -1 ) of each strain was spotted onto various media unless otherwise noted.…”

“…First strand cDNA, to be used as the qPCR template, was synthesized from 1 μg total RNA extracted as described previously ( Meng et al, 2017 ) with reverse transcriptase (TaKaRa, Dalian, China). qRT-PCR was carried out on a Real-Time PCR System (CFX Manager Software; Bio-Rad, Hercules, CA, United States) using a SYBR Green kit (SYBR Premix Ex Taq II; TaKaRa Dalian, China) according to manufacturer’s instructions.…”

G-protein subunit Gai in mitochondria, MrGPA1, affects conidiation, stress resistance, and virulence of entomopathogenic fungus Metarhizium robertsii

“…Fungal conidiation ability was evaluated as previously described (Meng et al 2017). Briefly, 30 ml of conidial suspension( 1 ´ 10 6 conidia /ml) was spread on PDA plate (35-mm diameter).…”

Chilling Stress Triggers VvAgo1-Mediated miRNA-Like RNA Biogenesis in Volvariella volvacea

“…The transcriptional machinery responsible for the synthesis of fungal miRNA-like RNAs (milRNAs) was first identified in Neurospora crassa (Yang et al, 2013). Various small RNA pathways involving Dicer and Ago genes central to the RNA interference (RNAi) machinery exist in the entomopathogenic fungus Metarhizium robertsii (Meng et al, 2017). The discovery of milRNAs has been reported mainly in pathogenic fungus, which represents their direct interactions with plants and animals (Bai et al, 2015;Wang et al, 2018;Zeng et al, 2018).…”