Novel miR-108 and miR-234 target juvenile hormone esterase to regulate the response of Plutella xylostella to Cry1Ac protoxin.

Insect hormones, such as juvenile hormone (JH), precisely regulate insect life-history traits. The regulation of JH is tightly associated with the tolerance or resistance to Bacillus thuringiensis (Bt). JH esterase (JHE) is a primary JH-specific metabolic enzyme which plays a key role in regulating JH titer. Here, we characterized a JHE gene from Plutella xylostella (PxJHE), and found it was differentially expressed in the Bt Cry1Ac resistant and susceptible strains. Suppression of PxJHE expression with RNAi increased the tolerance of P. xylostella to Cry1Ac protoxin. To investigate the regulatory mechanism of PxJHE, two target site prediction algorithms were applied to predict the putative miRNAs targeting PxJHE, and the resulting putative miRNAs were subsequently verified for their function targeting PxJHE using luciferase reporter assay and RNA immunoprecipitation. MiR-108 or miR-234 agomir delivery dramatically reduced PxJHE expression in vivo, whilst only miR-108 overexpression consequently increased the tolerance of P. xylostella larvae to Cry1Ac protoxin. By contrast, reduction of miR-108 or miR-234 dramatically increased PxJHE expression, accompanied by the decreased tolerance to Cry1Ac protoxin. Furthermore, injection of miR-108 or miR-234 led to developmental defects in P. xylostella, whilst injection of antagomir did not cause any obvious abnormal phenotypes. Our results indicated that miR-108 or miR-234 can be applied as potential molecular targets to combat P. xylostella and perhaps other lepidopteran pests, providing novel insights into miRNA-based integrated pest management.

Hsp70 and Hsp90 Elaborately Regulate RNAi Efficiency in Plutella xylostella.

Heat-shock proteins (HSPs) serve as molecular chaperones in the RNA interference (RNAi) pathway of eukaryotic organisms. In model organisms, Hsp70 and Hsp90 facilitate the folding and remodeling of the client protein Argonaute (Ago). However, the specific function of HSPs in the RNAi pathway of Plutella xylostella (L.) (Lepidoptera: Plutellidae) remains unknown. In this study, we identified and analyzed the coding sequences of PxHsc70-4 and PxHsp83 (also known as PxHsp90). Both PxHsc70-4 and PxHsp83 exhibited three conserved domains that covered a massive portion of their respective regions. The knockdown or inhibition of PxHsc70-4 and PxHsp83 in vitro resulted in a significant increase in the gene expression of the dsRNA-silenced reporter gene PxmRPS18, leading to a decrease in its RNAi efficiency. Interestingly, the overexpression of PxHsc70-4 and PxHsp83 in DBM, Sf9, and S2 cells resulted in an increase in the bioluminescent activity of dsRNA-silenced luciferase, indicating a decrease in its RNAi efficiency via the overexpression of Hsp70/Hsp90. Furthermore, the inhibition of PxHsc70-4 and PxHsp83 in vivo resulted in a significant increase in the gene expression of PxmRPS18. These findings demonstrated the essential involvement of a specific quantity of Hsc70-4 and Hsp83 in the siRNA pathway in P. xylostella. Our study offers novel insights into the roles played by HSPs in the siRNA pathway in lepidopteran insects.

Functional characteristics of Dicer genes in Plutella xylostella.

BACKGROUND: Dicer is an endonuclease that belongs to the RNase III family and can specifically recognize and cleave double-stranded RNA (dsRNA). In most insects, there are two Dicer genes, Dicer-1 (Dcr-1) and Dicer-2 (Dcr-2), which are involved in the micro-RNA and small-interfering RNA pathways in many species, respectively. The function of Dicer in Plutella xylostella remains unknown. RESULTS: The full-length open reading frames of P. xylostella Dicer-1 (PxDcr-1) and Dicer-2 (PxDcr-2) were cloned and sequenced. Dcr-1 and Dcr-2 proteins shared similar structural domains with the Dicer-Partner Binding Domain (Dicer-PBD) and the double-strand RNA binding domain (dsRBD) present only in Dcr-1. The phylogenetic trees showed that lepidopteran Dcr-1s or Dcr-2s clustered in one branch, with PxDcr-1 in the basal position and PxDcr-2 closest to Plodia interpunctella Dicer. Two homozygous knockout lines, ΔPxDcr-1 and ΔPxDcr-2, were obtained by using the CRISPR-Cas9 technique. The ΔPxDcr-1 strain exhibited a high mortality rate, a low eclosion rate, a low egg-laying rate, a low hatching rate, and a shriveled ovariole without mature eggs. The ΔPxDcr-2 strain showed no significant difference from the wild-type in terms of survival, development and reproduction, but the RNA interference (RNAi) efficiency caused by dsRNA was significantly reduced. CONCLUSION: These findings demonstrate the involvement of PxDcr-1 in the development and reproduction of P. xylostella, specifically in the formation of ovarioles and eggs, and PxDcr-2 is indispensable for RNAi. These findings shed light on the function of Dcr-1 and Dcr-2 in Lepidoptera. © 2023 Society of Chemical Industry.

Novel-miR-310 mediated response mechanism to Cry1Ac protoxin in Plutella xylostella (L.).

The diamondback moth (DBM), Plutella xylostella (L.), has evolved resistance to multiple insecticides including Bacillus thuringiensis (Bt). ATP-binding cassette (ABC) transporters are a class of transmembrane protein families, involved in multiple physiological processes and pesticide resistances in insects. However, the role and regulatory mechanism of ABC transporter in mediating the response to Bt Cry1Ac toxin remain unclear. Here, we characterized a MAPK signaling pathway-enriched ABCG subfamily gene PxABCG20 from DBM, and found it was differentially expressed in the Cry1Ac-resistant and Cry1Ac-susceptible strains. RNAi knockdown of PxABCG20 increased the tolerance of DBM to Cry1Ac protoxin. To explore the regulatory mechanism of PxABCG20 expression, we predicted the potential miRNAs targeting PxABCG20 using two target prediction algorithms. Luciferase reporter assay confirmed that novel-miR-310 was able to down-regulate PxABCG20 expression in HEK293T cells. Furthermore, injection of novel-miR-310 agomir markedly inhibited PxABCG20 expression, resulting in increased tolerance to Cry1Ac protoxin in susceptible strain, while injection of novel-miR-310 antagomir markedly induced the expression of PxABCG20, leading to decreased tolerance to Cry1Ac protoxin. Our work provides theoretical basis for exploring novel targets for the DBM response to Cry1Ac toxin and expands the understanding of miRNA role in mediating the susceptibility of insect pest to Cry1Ac toxin.

Profiling of MicroRNAs in Midguts of Plutella xylostella Provides Novel Insights Into the Bacillus thuringiensis Resistance.

The diamondback moth (DBM), Plutella xylostella, one of the most destructive lepidopteran pests worldwide, has developed field resistance to Bacillus thuringiensis (Bt) Cry toxins. Although miRNAs have been reported to be involved in insect resistance to multiple insecticides, our understanding of their roles in mediating Bt resistance is limited. In this study, we constructed small RNA libraries from midguts of the Cry1Ac-resistant (Cry1S1000) strain and the Cry1Ac-susceptible strain (G88) using a high-throughput sequencing analysis. A total of 437 (76 known and 361 novel miRNAs) were identified, among which 178 miRNAs were classified into 91 miRNA families. Transcripts per million analysis revealed 12 differentially expressed miRNAs between the Cry1S1000 and G88 strains. Specifically, nine miRNAs were down-regulated and three up-regulated in the Cry1S1000 strain compared to the G88 strain. Next, we predicted the potential target genes of these differentially expressed miRNAs and carried out GO and KEGG pathway analyses. We found that the cellular process, metabolism process, membrane and the catalytic activity were the most enriched GO terms and the Hippo, MAPK signaling pathway might be involved in Bt resistance of DBM. In addition, the expression patterns of these miRNAs and their target genes were determined by RT-qPCR, showing that partial miRNAs negatively while others positively correlate with their corresponding target genes. Subsequently, novel-miR-240, one of the differentially expressed miRNAs with inverse correlation with its target genes, was confirmed to interact with Px017590 and Px007885 using dual luciferase reporter assays. Our study highlights the characteristics of differentially expressed miRNAs in midguts of the Cry1S1000 and G88 strains, paving the way for further investigation of miRNA roles in mediating Bt resistance.