Gram-negative binding proteins (GNBPs), also known as β-1,3-glucan recognition proteins, represent a class of crucial pattern recognition receptors (PRRs) in insects and play pivotal roles in the innate immune response. This study aimed to systematically identify members of the PxGNBP gene family in the diamondback moth (Plutella xylostella), analyze their structural characteristics and expression patterns, as well as screen and validate key target genes. The findings are expected to reveal the innate immune mechanisms and evolutionary adaptations of P. xylostella in response to pathogenic infection, thereby providing theoretical foundations and potential targets for the development of novel biological control.

Based on the whole-genome data of P. xylostella, members of the PxGNBP gene family were identified. Bioinformatic approaches were comprehensively employed to analyze their structural characteristics and evolutionary relationships, and AlphaFold3 was used to predict their three-dimensional structures. In addition, combined with public transcriptome data and quantitative real-time polymerase chain reaction (RT-qPCR) technology, the expression patterns of these family members in different tissues and post-infection with Beauveria bassiana and Metarhizium anisopliae were detected. Recombinant M. anisopliae strains carrying pSilent-PxGNBP3 were constructed. The expression levels of PxGNBP3 and downstream antimicrobial peptide genes post-infection were determined via RT-qPCR, and the pathogenicity of different strains against P. xylostella was evaluated using bioassays.

A total of 10 PxGNBP members were identified in P. xylostella. Among them, PxβGRP4 is located on chromosome 22 and belongs to the glucanase subfamily, while the remaining 9 members are located on chromosome 29 and belong to the PRR subfamily. Phylogenetic and chromosome location analyses suggested the occurrence of tandem duplication events within this gene family. Conserved motif analysis indicated that the N-terminal domain of PxGNBP exhibited lower conservation compared to the C-terminal domain. Except for PxβGRP4, the key catalytic sites of glucanase in other members were mutated. Three-dimensional structure predictions revealed that all members, except PxβGRP4 and PxβGRP3, possessed the typical GNBP protein structure; the C-terminus of PxβGRP3 contained a structural fragment that was similar but not identical to Carbohydrate-binding module 39 (CBM39). Expression profile analysis demonstrated that most members exhibited a time-series expression pattern of first increasing and then decreasing after infection with the two fungi. RNA interference (RNAi) assays showed that the recombinant M. anisopliae strains could effectively suppress the expression of PxGNBP3, leading to a significant reduction in antimicrobial peptide expression levels and a decrease in host survival rate. Moreover, the virulence of recombinant strains was significantly higher than that of the wild-type strain and enhanced with increasing concentration.

Ten members of the GNBP gene family were identified in P. xylostella, with PxβGRP3 and PxGNBP3-2 showing structural specificity. This gene family exhibited a time-series regulatory expression pattern in response to fungal infection. In vivo functional validation of PxGNBP3 via RNAi was successfully achieved using the constructed recombinant M. anisopliae strains. The results provide important insights for elucidating the innate immune mechanisms of P. xylostella and developing novel targets for biological control.

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.