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MicroRNA Targets PAP1 to Mediate Melanization in Plutella xylostella (Linnaeus) Infected by Metarhizium anisopliae
Zhantao Zhang1, Fengliang Jin1, Junlin Huang1
1National Key Laboratory of Green Pesticide, College of Plant Protection, South China Agricultural University, Guangzhou 510642, China.
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.
Insights
MicroRNAs regulate insect immunity. This study identifies pxy-miR-965-5p as a key regulator of the prophenoloxidase cascade in diamondback moths, impacting their immune response to fungal infection.
Area of Science:
- Molecular Biology
- Insect Pathology
- Genomics
Background:
- MicroRNAs (miRNAs) are crucial regulators of gene expression with roles in immunity.
- The diamondback moth (Plutella xylostella) is a significant agricultural pest.
- Limited understanding exists regarding miRNA involvement in P. xylostella immunity.
Purpose of the Study:
- Identify miRNAs targeting prophenoloxidase-activating protease1 (PAP1) in P. xylostella.
- Investigate the role of identified miRNAs in the phenoloxidase (PO) cascade and melanization.
- Explore potential long non-coding RNA (lncRNA) interactions with key miRNAs.
Main Methods:
- Whole transcriptome resequencing of P. xylostella infected with Metarhizium anisopliae.
- Bioinformatic screening for miRNAs targeting PAP1.
- Luciferase reporter assays to confirm miRNA-mRNA interactions.
- miRNA mimic and inhibitor experiments in P. xylostella larvae.
- LncRNA identification and interference assays using antisense oligonucleotides (ASOs).
Main Results:
- Seven miRNAs were screened, with pxy-miR-279a-3p and pxy-miR-965-5p confirmed to target PAP1.
- pxy-miR-965-5p mimic suppressed PAP1 expression, reduced PO activity, and increased larval mortality.
- pxy-miR-965-5p inhibitor increased PAP1 expression and PO activity, decreasing larval mortality.
- Four lncRNAs were identified; silencing MSTRG.7100.1 and MSTRG.22113.1 increased pxy-miR-965-5p expression.
Conclusions:
- pxy-miR-965-5p plays a significant role in regulating the immune response of P. xylostella to M. anisopliae infection.
- This miRNA targets PAP1, influencing the PO cascade and larval survival.
- Identified lncRNAs may modulate pxy-miR-965-5p activity, suggesting complex regulatory networks.
- Findings provide a basis for developing novel biological control strategies targeting pest immunity.
