MicroRNA Targets PAP1 to Mediate Melanization in Plutella xylostella (Linnaeus) Infected by Metarhizium anisopliae

Abstract

MicroRNAs (miRNAs) play a pivotal role in important biological processes by regulating post-transcriptional gene expression and exhibit differential expression patterns during development, immune responses, and stress challenges. The diamondback moth causes significant economic damage to crops worldwide. Despite substantial advancements in understanding the molecular biology of this pest, our knowledge regarding the role of miRNAs in regulating key immunity-related genes remains limited. In this study, we leveraged whole transcriptome resequencing data from Plutella xylostella infected with Metarhizium anisopliae to identify specific miRNAs targeting the prophenoloxidase-activating protease1 (PAP1) gene and regulate phenoloxidase (PO) cascade during melanization. Seven miRNAs (pxy-miR-375-5p, pxy-miR-4448-3p, pxy-miR-279a-3p, pxy-miR-3286-3p, pxy-miR-965-5p, pxy-miR-8799-3p, and pxy-miR-14b-5p) were screened. Luciferase reporter assays confirmed that pxy-miR-279a-3p binds to the open reading frame (ORF) and pxy-miR-965-5p to the 3' untranslated region (3' UTR) of PAP1. Our experiments demonstrated that a pxy-miR-965-5p mimic significantly reduced PAP1 expression in P. xylostella larvae, suppressed PO activity, and increased larval mortality rate. Conversely, the injection of pxy-miR-965-5p inhibitor could increase PAP1 expression and PO activity while decreasing larval mortality rate. Furthermore, we identified four LncRNAs (MSTRG.32910.1, MSTRG.7100.1, MSTRG.6802.1, and MSTRG.22113.1) that potentially interact with pxy-miR-965-5p. Interference assays using antisense oligonucleotides (ASOs) revealed that silencing MSTRG.7100.1 and MSTRG.22113.1 increased the expression of pxy-miR-965-5p. These findings shed light on the potential role of pxy-miR-965-5p in the immune response of P. xylostella to M. anisopliae infection and provide a theoretical basis for biological control strategies targeting the immune system of this pest.

Keywords: diamondback moth; melanization; non-coding RNAs; pest management.

Figure 1

The infection of P. xylostella by M. anisopliae. (A) The phenomena of infection. (B) Determination of PO activity. “Control” indicates the group treated by conidia germinating medium, and “Ma” indicates the group infected by M. anisopliae. The column represented the PO activity, and data in the figure are mean ± SE (LSD and Duncan analysis: α = 0.05, n = 3, letters on the columns indicate significant differences among groups, p < 0.05).

Figure 2

PAP1 activated P. xylostella melanization in vitro. (A) The mode of PAP1 and IEGR mutant PAP1_Xa. (B) The cleavage of PAP1_Xa by Factor Xa, monoclonal antibody 6 × His was used as the primary antibody, marker was on the left; protein PAP1_Xa was on the line 57.3 kDa and protein activated PAP1_Xa after cleaved was on line 29.4 kDa. (C) The enzyme activity of PAP1. The column represents the enzyme activity, data in the figure are mean ± SE (two-tailed Student’s t-tests: α = 0.05, n = 3, asterisks indicate significant differences between treatment and control groups, * p < 0.05, ** p < 0.01, *** p < 0.001, NS = no significant differences).

Figure 3

The miRNA target prediction and verification of PAP1. (A) Sites on PAP1 targeted by miRNAs. 5′ UTR, 5′ untranslated region; ORF, Open Reading Frame; 3′ UTR, 3′ untranslated region. (B) The dual luciferase reporter assay of miRNAs to PAP1. The columns represent the RFU (hRluc/hluc+) fold change in miRNA mimic treatment groups compared to negative control groups, data in the figure are mean ± SE (two-tailed Student’s t-tests: α = 0.05, n = 3, asterisks indicate significant differences between treatment and control groups, *** p < 0.001, NS = no significant differences).

Figure 4

The expression patterns of PAP1 and miRNA. (A) The expression patterns in different tissues. The column represented the relative expression of PAP1, pxy-miR-965-5p, and pxy-miR-279a-3p, data in the figure are mean ± SE (LSD and Duncan analysis: α = 0.05, n = 3, letters on the columns indicated significant differences among different tissues, p < 0.05). (B) The expression patterns after M. anisopliae infection. The columns represent the relative expression fold change in treatment groups compared to control groups; data in the figure are mean ± SE (two-tailed Student’s t-tests: α = 0.05, n = 3, asterisks indicate significant differences between treatment and control groups, * p < 0.05, ** p < 0.01, *** p < 0.001, NS = no significant differences).

Figure 5

The expression of PAP1 in P. xylostella under miRNA treatments. (A) The expression of PAP1 under pxy-miR-965-5p treatments. Ma, the group infected by M. anisopliae; Ma + NC, the group infected by M. anisopliae after mimics negative control injection; Ma + mimic, the group infected by M. anisopliae after pxy-miR-965-5p mimic injection; Ma + inhibitor, the group infected by M. anisopliae after pxy-miR-965-5p inhibitor injection. The columns represent the relative expression of PAP1, data in the figure are mean ± SE (LSD and Duncan analysis: α = 0.05, n = 3, letters on the columns indicate significant differences among groups, p < 0.05). (B) The expression of PAP1 under pxy-miR-279a-3p treatments. Ma, the group infected by M. anisopliae; Ma + NC, the group infected by M. anisopliae after mimics negative control injection; Ma + mimic, the group infected by M. anisopliae after pxy-miR-279a-3p mimic injection; Ma + inhibitor, the group infected by M. anisopliae after pxy-miR-279a-3p inhibitor injection. The columns represent the relative expression of PAP1, data in the figure are mean ± SE (LSD and Duncan analysis: α = 0.05, n = 3, NS = no significant differences).

Figure 6

Survival rates and PO activity of P. xylostella under miRNA treatments. (A) Survival rates of P. xylostella under miRNA treatments. Ma, the group infected by M. anisopliae; Ma + NC, the group infected by M. anisopliae after mimics negative control injection; Ma + pxy-miR-965-5p mimic, the group infected by M. anisopliae after pxy-miR-965-5p mimic injection; Ma + pxy-miR-965-5p inhibitor, the group infected by M. anisopliae after pxy-miR-965-5p inhibitor injection. The curve represents the survival rates (Log-rank test: α = 0.05, n = 100, letters on the columns indicate significant differences among groups, p < 0.05). (B) PO activity of P. xylostella under miRNA treatments. (B) PO activity of P. xylostella under miRNA treatments. Ma, the group infected by M. anisopliae; Ma + NC, the group infected by M. anisopliae after mimics negative control injection; Ma + mimic, the group infected by M. anisopliae after pxy-miR-965-5p mimic injection; Ma + inhibitor, the group infected by M. anisopliae after pxy-miR-965-5p inhibitor injection. The columns represent the PO activity, data in the figure are mean ± SE (LSD and Duncan analysis: α = 0.05, n = 3, letters on the columns indicate significant differences among groups, p < 0.05).

Figure 7

The dual luciferase reporter assay of LncRNA to pxy-miR-965-5p. The column represented the RFU (hRluc/hluc+) fold change in miRNA mimic treatment groups compared to negative control groups, data in the figure are mean ± SE (two-tailed Student’s t-tests: α = 0.05, n = 3, asterisks indicate significant differences between treatment and control groups, ** p < 0.01).

Figure 8

The expression of LncRNA and miRNA in P. xylostella after ASO injection. (A) The expression of LncRNA after ASO injection. Ma, the group infected by M. anisopliae; Ma + NC, the group infected by M. anisopliae after ASO negative control injection; Ma + ASO, the group infected by M. anisopliae after ASO injection. The columns represent the relative expression of LncRNA; data in figure are mean ± SE (LSD and Duncan analysis: α = 0.05, n = 3, letters on the columns indicate significant differences among groups, p < 0.05). (B) The expression of pxy-miR-965-5p after ASO injection. Ma, the group infected by M. anisopliae; Ma + NC, the group infected by M. anisopliae after ASO negative control injection; Ma + ASO, the group infected by M. anisopliae after ASO injection. The columns represent the relative expression of LncRNA; data in the figure are mean ± SE (LSD and Duncan analysis: α = 0.05, n = 3, letters on the columns indicate significant differences among groups, p < 0.05, NS = no significant differences).