Genome-Wide Identification and Analysis of Nilaparvata lugens microRNAs during Challenge with the Entomopathogenic Fungus Metarhizium anisopliae

Abstract

The resistance of the notorious rice pest Nilaparvata lugens to many insecticides has caused significant concerns. Our previous study demonstrated that the fungus Metarhizium anisopliae CQMa421 shows great potential for the control of this pest, but the interactions between them are still unclear. Thus, we further investigated fungal infection-related microRNAs (miRNAs) in N. lugens during M. anisopliae CQMa421 challenge using Illumina sequencing. In this study, we constructed twenty-four small RNA libraries over different time courses (i.e., 4 h, 8 h, 16 h, and 24 h). A total of 478.62 M clean reads were collected, with each sample producing more than 13.37 M reads, after the removal of low-quality reads. We identified 2324 miRNAs and their 11,076 target genes within the twenty-four libraries by bioinformatics analysis. Differentially expressed miRNAs (DEmiRNAs), including 58 (32 upregulated vs. 26 downregulated), 62 (30 upregulated vs. 32 downregulated), 126 (71 upregulated vs. 55 downregulated), and 109 (40 upregulated vs. 69 downregulated) DEmiRNAs were identified at 4 h, 8 h, 16 h, and 24 h post-infection, respectively. We further conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to predict the functions of all target genes of DEmiRNAs. These DEmiRNAs targets identified during 24 h of infection were primarily involved in energy metabolism, lysine degradation, the FoxO signaling pathway, ubiquitin-mediated proteolysis, the mRNA surveillance pathway, and the MAPK signaling pathway. Taken together, our results provide essential information for further study of the interactions between the entomopathogenic fungus M. anisopliae and N. lugens at the posttranscriptional level.

Keywords: Metarhizium anisopliae; Nilaparvata lugens; entomopathogenic fungus; fungal infection; microRNAs; pest control.

Figure 1

The length distribution and nucleotide bias of unique small RNA reads in the libraries of N. lugens. (A) The total length distribution of N. lugens small RNAs in the twenty-four libraries (4 h, 8 h, 16 h, and 24 h) generated after M. anisopliae infection; (B) the first nucleotide bias at each position among miRNAs of different lengths in the twenty-four libraries.

Figure 2

The volcano plot and numbers of differentially expressed miRNAs (DEmiRNAs) identified after M. anisopliae infection. (A) The volcano plot of DEmiRNAs at 4 h post-challenge; (B) the volcano plot of DEmiRNAs at 8 h post challenge; (C) the volcano plot of DEmiRNAs at 16 h post-challenge; (D) the volcano plot of DEmiRNAs at 24 h post-challenge; and (E) the Venn diagram of DEmiRNAs after M. anisopliae infection at 4, 8, 16, and 24 h.

Figure 3

The GO categories of N. lugens DEmiRNAs identified during M. anisopliae infection. (A) The GO categories of N. lugens DEmiRNAs at 4 h post M. anisopliae infection; (B) the GO categories of N. lugens DEmiRNAs at 8 h post M. anisopliae infection; (C) the GO categories of N. lugens DEmiRNAs at 16 h post M. anisopliae infection, and (D) the GO categories of N. lugens DEmiRNAs at 24 h post M. anisopliae infection.

Figure 4

The enrichment and dispersion of differentially expressed miRNAs in KEGG pathways after M. anisopliae infection. (A) The identified pathways of DEmiRNAs after 4 h of M. anisopliae infection; (B) the identified pathways of DEmiRNAs after 8 h of M. anisopliae infection; (C) the identified pathways of DEmiRNAs after 16 h of M. anisopliae infection; and (D) the identified pathways of DEmiRNAs after 24 h of M. anisopliae infection. The asterisks indicate significant differences according to a p value < 0.05.

Figure 5

The validation of RNA sequencing and RT-qPCR results for target genes. Three replicates were performed for each miRNA, and error bars represent the mean ± SE.