The Role of miR-6497-x in Regulating the Reaction of Plutella xylostella to Fungal Infection

The phenoloxidase (PO) activation system is essential for insect innate immunity, particularly in pathogen defense, with prophenoloxidase-activating protease (PAP) being a key component that directly activates prophenoloxidase (proPO). However, research on these components in the insect PO activation system remains limited.

The objectives of this study are to explore the regulatory role of microRNA (miRNA) in the phenoloxidase activation system of Plutella xylostella infected by Metarhizium anisopliae, and to provide new targets and approaches for pest control.

Bioinformatics was used to identify miRNAs targeting specific mRNAs. Real-time quantitative PCR (qRT-PCR) was employed to assess the transcription levels of miRNAs, PAP2, and PAP3 of P. xylostella at different time points post-infection with M. anisopliae (1×10⁶ CFU/mL). The regulatory effects of miRNAs on PAP2 and PAP3 were evaluated using a dual-luciferase system. Moths were injected with miRNA mimics or inhibitors and infected with M. anisopliae 12 h later. The expression levels of PAP2 and PAP3 were measured by qRT-PCR, while mortality and PO activity were also assessed.

miR-6497-x, miR-8545-x, novel-m0313-3p, and novel-m0592-5p target PAP3, while novel-m0042-5p, pxy-miR-2756-3p, and miR-9215-x target PAP2. A negative regulatory relationship between miRNAs and their target genes was observed at 24 and 48 h post-infection with M. anisopliae. In vitro experiments confirmed that miR-6497-x, novel-m0313-3p, and novel-m0592-5p negatively regulated PAP3, while miR-9215-x significantly downregulated PAP2. In vivo injection of miR-6497-x mimic led to decreased PAP3 expression, increased larval mortality, and reduced PO activity within 12 to 48 h post-infection. Conversely, injection of miR-6497-x inhibitor resulted in upregulated PAP3 expression, decreased larval mortality, and increased PO activity. However, overexpression or inhibition of miR-9215-x did not significantly affect PAP2 expression, larval mortality, or PO activity compared to the control group during the same period post-infection.

The miR-6497-x targeting PAP3 was screened and identified. Both in vitro and in vivo experiments confirm that miR-6497-x negatively regulates PAP3, thereby affecting the PO cascade. miR-6497-x plays a crucial role in modulating the immune defense of P. xylostella against M. anisopliae infection, which will provide a theoretical basis for biological control strategies targeting pest immune systems.