Abstract
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.
Highlights
Bacillus thuringiensis (Bt), a class of spore-forming gram-positive bacterium that can produce different insecticidal crystal proteins (Cry and Cyt toxins), has been widely used as an entomopathogen for pest control (Wu et al, 2008; Raymond et al, 2010; Palma et al, 2014)
To analyze the role of miRNAs in the Bt resistance of diamondback moth (DBM), we performed the deep sequencing for identification and characterization of miRNAs in the larval midguts of the Cry1S1000 and G88 strains
The clean reads were aligned with Silva, GtRNAdb, Rfam, and Repbase databases and mapped to the reference genome using the Bowtie package
Summary
Bacillus thuringiensis (Bt), a class of spore-forming gram-positive bacterium that can produce different insecticidal crystal proteins (Cry and Cyt toxins), has been widely used as an entomopathogen for pest control (Wu et al, 2008; Raymond et al, 2010; Palma et al, 2014). Bt Cry toxin has specific control efficiency on lepidopteran pests, and the mode of action involves toxin solubilization, proteolytic activation, interaction with midgut proteins of insects, formation of a prepore oligomeric structure, facilitation of the insertion into cell membrane, and creation of an ionic pore that kills midgut cells (Bravo et al, 2011). More than 22 cases of field-evolved Bt resistance in several insect species have been documented, potentially reducing the control efficacy of Bt toxins toward agricultural pests (Janmaat and Myers, 2003; Tabashnik and Carrière, 2017, 2019; CallesTorrez et al, 2019). To delay the resistance evolution, it is urgent to understand the mechanisms causing Bt resistance and provide novel targets for pest control
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